Gene expression-based biomarker for the detection and monitoring of bronchial premalignant lesions

ABSTRACT

Disclosed herein are assays and methods for the identification of premalignant lesions, as well as methods of determining the likelihood that such premalignant lesions will progress to lung cancer. Also disclosed are methods and assays that are useful for monitoring the progression of premalignant lesions to lung cancer. The assays and methods disclosed herein provide minimally invasive means of accurately detecting and monitoring the presence or absence of premalignant lesions, thus providing novel insights into the earliest stages of lung cancer and facilitating early detection and early intervention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 15/644,721, filed Jul. 7, 2017, which claims the benefit of U.S. Provisional Application No. 62/360,218, filed on Jul. 8, 2016, the contents of which are hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Lung cancer (LC) is the leading cause of cancer death in the United States. The molecular events preceding the onset of LC and the progression of premalignant lesions (PMLs) to lung cancer are poorly understood. This is due in part to the lack of reliable biomarkers which complicates the study of such lesions. Currently there are no molecular tests to identify PMLs or describe their changes over time. The only technology that is able to visualize and sample premalignant lesions is auto-fluorescent bronchoscopy, which is limited in sensitivity and is not in widespread clinical use.

Needed are novel biomarkers, methods and assays that are capable of facilitating the evaluation of PMLs. Suspicious lesions on chest computed tomography (CT) scans typically prompt bronchoscopic evaluation, which is also limited by varying diagnostic yields. Moreover, negative bronchoscopies prove a clinical dilemma, whereby the need to provide a diagnostic answer is countered by the invasiveness of follow-up studies.

A previously reported biomarker, PERCEPTA® (Veracyte Inc.), has demonstrated the potential benefit of employing a bronchial gene expression-based classifier on a sub-set of patients with non-diagnostic bronchoscopies, through modifying risk stratification of patients. However, this biomarker has demonstrated greatest benefit amongst those with a moderate pre-test probability with modest overall sensitivities. The employment of a novel pre-malignancy marker would complement the PERCEPTA® biomarker in this sub-set of patients, facilitating the identification of those patients that would be at high risk for PML progression.

Also needed are new biomarkers, methods and assays for use in lung cancer screening assays and the early detection of PMLs. A recent large randomized controlled trial has led to the recent endorsement of annual lung cancer screening with low dose CT for asymptomatic patients that are at higher lung cancer risk. This has created a large volume of chest CTs, whose performance is marred by the high rate of false positive results. It is anticipated that this will lead to a large need for invasive procedures for benign disease. A pre-malignancy biomarker could complement the diagnostic work up of lesions identified through screening, which are typically more complicated since such lesions identified on screening are usually smaller and more complex. Additionally, patient screening eligibility is based solely on epidemiological and demographic considerations, which still vary between different proposed guidelines. This leads to varying referral patterns and missed opportunities to screen a large proportion of those patients with high risk that do not meet dictated criteria. The availability of biomarkers, methods and assays for the detection of PMLs would overcome this challenge by facilitating the identification of pre-malignancy-associated changes and risk of progression, would provide a first step to identifying molecular risk factors for lung cancer, and would identify those patients who would benefit from CT screening. Such biomarkers would also be useful for patent risk stratification, which would assist in the identification of those patients that may benefit from additional screening of those patients harboring premalignant molecular alterations, which could in turn inform future decision making.

The limited understanding of the mechanisms involved in transforming PMLs into LC has restricted the ability to intervene in these processes, making the identification of chemoprevention agents difficult in view of the challenges involved in discerning premalignant phenotypes through currently available means. Furthermore, clinical trials in this space are exceedingly difficult given the long duration required to detect significant outcome benefits. Accordingly, biomarkers, assays and methods that are reflective of pre-malignancy would facilitate “smart” patient enrollment for trials and would allow accounting for molecular heterogeneity involved in random patient recruitment in such trials.

SUMMARY OF THE INVENTION

The present inventions provide insight into the mechanisms that are involved in the transformation or progression of premalignant bronchial lesions into lung cancer. Provided herein are novel biomarkers, methods and assays that are useful in lung cancer screening and the early detection of premalignant lesions (PMLs). The biomarkers, methods and assays of the present invention also facilitate the monitoring of PMLs and their progression or regression over time. Advantageously, the assays and methods disclosed herein may be rapidly performed in a non-invasive or minimally-invasive manner, providing objective results, contributing to the identification and monitoring of subjects that are suspected of having PMLs, facilitating the clinical decision making of the treatment of such subjects and informing clinical trial recruitment efforts.

In certain aspects, the biomarkers, methods and assays disclosed herein may be assessed or performed on a biological sample that is obtained from a subject at a site that is distal to the suspected site of the premalignant bronchial lesion. For example, in certain embodiments, the assays and methods of determining the presence of PMLs or cancer in the lungs may be performed by determining the expression of one or more genes in nasal or buccal epithelial cells and/or tissues. Similarly, such assays and methods may be performed by determining the expression of one or more genes in the subject's peripheral blood cells. In certain aspects, the biomarkers, methods and assays disclosed herein may be assessed or performed on, or additionally include, a biological sample that is obtained from a subject with a positive result in an imaging study (e.g., chest X-ray, CT scan, etc.). In some aspects, the methods and assays disclosed herein can comprise a step of performing an imaging study. In certain aspects, the biomarkers, methods and assays disclosed herein may be assessed or performed on, or additionally include, a biological sample that is obtained from a subject with a positive result in an imaging study (e.g., chest X-ray, CT scan, etc.) to confirm or rule out the positive result. In some aspects, the methods or assays disclosed herein are used to determine whether a positive result in an imaging study warrants a further invasive procedure (e.g., bronchoscopy), chemoprophylaxis, and/or chemotherapy.

In some embodiments, methods and assays disclosed herein may be assessed or performed on a biological sample that is obtained from a subject at a suspected site of a PML (e.g., premalignant bronchial lesion). In some embodiments, the suspected site is identified as having abnormal fluorescent during auto-fluorescence bronchoscopy, although the method of identifying the suspected site is not limited. In some embodiments, the methods and assays disclosed herein may be performed on a biopsy of a suspected PML as an alternative to, or in addition to, a histological examination of the biopsy.

In certain aspects, disclosed herein are methods of determining the presence or absence of a premalignant lesion in a subject. Such methods comprise the steps of: (a) measuring a biological sample comprising airway epithelial cells of the subject for expression of one or more genes; and (b) comparing the expression of the one or more genes to a control sample of those genes from individuals without premalignant lesions; wherein the one or more genes are selected from the group consisting of genes in Table 3, and wherein differential expression of the subject's one or more genes relative to the control sample is indicative of the presence of a premalignant lesion in the subject. Similarly, in certain embodiments, non-differential expression of the subject's one or more genes relative to the control sample is indicative of the absence of a premalignant lesion in the subject.

Also disclosed herein are methods of determining the likelihood that a premalignant lesion in a subject will progress to lung cancer. In certain aspects, such methods comprise the steps of: (a) measuring a biological sample comprising airway epithelial cells of the subject for expression of one or more genes; and (b) comparing the expression of the one or more genes to a control sample of those genes from individuals with lung cancer; wherein the one or more genes are selected from the group consisting of genes in Table 3, and wherein differential expression of the subject's one or more genes relative to the control sample is indicative of a low likelihood of the premalignant lesion progressing to lung cancer. In some embodiments, non-differential expression of the subject's one or more genes relative to the control sample is indicative of a high likelihood of the premalignant lesion progressing to lung cancer.

In certain embodiments, also disclosed herein are methods of monitoring whether a premalignant lesion will progress to lung cancer in a subject. Such methods comprise subjecting a biological sample comprising airway epithelial cells of the subject to a gene expression analysis, wherein the gene expression analysis comprises comparing gene expression levels of one or more genes selected from the group of genes in Table 3 to the expression levels of a control sample of those genes from individuals with cancer, and wherein differential expression of the subject's one or more genes relative to the control sample is indicative of a lack of progression of the premalignant lesion to lung cancer. Similarly, in certain aspects non-differential expression of the subject's one or more genes relative to the control sample is indicative of progression of the premalignant lesion to lung cancer.

In yet other embodiments, also disclosed herein are methods of determining the presence of a premalignant lesion in a subject comprising the steps of: (a) measuring a biological sample comprising airway epithelial cells of the subject for expression of one or more genes; and (b) comparing the expression of the one or more genes to a control sample of those genes obtained from individuals without premalignant lesions; wherein the one or more genes are selected from the group of genes in at least one pathway in Dataset 2, and wherein differential expression of the subject's one or more genes relative to the control sample is indicative of the presence of a premalignant lesion in the subject. In some embodiments, non-differential expression of the subject's one or more genes relative to the control sample is indicative of the absence of a premalignant lesion in the subject.

In certain aspects of any of the foregoing methods, at least two genes, at least five genes, at least ten genes, at least twenty genes, at least thirty genes, at least forty genes, at least fifty genes, at least one hundred genes, at least two hundred genes or at least two hundred and eighty genes are measured. In some embodiments of the foregoing methods, the one or more genes comprise those genes associated with a pathway identified in Dataset 2.

In some embodiments of any of the foregoing methods the airway epithelial cells comprise bronchial epithelial cells. In certain aspects, such bronchial epithelial cells are obtained by brushing the bronchi walls of the subject. In certain aspects of any of the foregoing methods, the airway epithelial cells comprise nasal epithelial cells. In certain aspects of any of the foregoing methods, the airway epithelial cells comprise buccal epithelial cells. In still other embodiments of the present inventions, the airway epithelial cells do not comprise bronchial epithelial cells. In some embodiments, the airway epithelial cells are obtained from a suspected PML site (e.g., abnormal fluorescing areas during auto-fluorescence bronchoscopy).

In certain aspects, the methods disclosed herein are performed with, or further comprise assessing or determining one or more of the subject's secondary factors that affect the subject's risk for having or developing lung cancer. For example, in some embodiments, one or more secondary factors are selected from the group consisting of advanced age, smoking status, the presence of a lung nodule greater than 3 cm on CT scan and time since quitting smoking. In certain embodiments of the foregoing methods, expression of the one or more genes is determined using a quantitative reverse transcription polymerase chain reaction, a bead-based nucleic acid detection assay or an oligonucleotide array assay.

The foregoing methods are useful for predicting or monitoring the progression of PMLs to lung cancer. For example, a lung cancer selected from the group consisting of adenocarcinoma, squamous cell carcinoma, small cell cancer or non-small cell cancer.

In some embodiments, the one or more genes comprise mRNA and/or microRNA. In some embodiments, the differential expression is determined by reverse transcribing one or more RNAs of the one or more genes into cDNA in vitro. In some aspects, the one or more genes comprise cDNA. In yet other embodiments, the one or more genes are labeled prior to the measuring.

The above discussed, and many other features and attendant advantages of the present inventions will become better understood by reference to the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 represents a flow diagram depicting the design of the study used in the Examples. Depicted is the use of bronchial brushings collected from subjects with (red, n=50) and without (gray, n=25) PMLs from the BCCA as part of the BC-LHS for differential gene expression/pathway analysis and for biomarker development. Independent human and mouse bronchial biopsies and biopsy cell cultures were used to validate these findings via mitochondrial enumeration, bioenergetics, and immunohistochemistry (left panel). Biomarker development was conducted by splitting samples from the BC-LHS into a discovery (n=58) and a validation set (Validation 1, n=17) (right panel). The discovery set was used to create the gene expression-based biomarker to detect the presence of PMLs in the airway field of injury. The biomarker was tested on the BC-LHS validation set and an external validation set (bottom) from RPCI (Validation 2, n=28 matched time point pairs, stable/progressing pairs in yellow and regressing pairs in blue).

FIG. 2 shows an unsupervised hierarchal clustering of genes associated with the presence of premalignant lesions. Residual gene expression of the 280 genes differentially expressed between subjects with PMLs (red) and without PMLs (gray). Top color bars represent the worst biopsy histological grade observed during bronchoscopy and genomically-derived smoking status of the subjects. The 14 genes in the KEGG oxidative phosphorylation pathway are indicated in cyan. The residual values after adjusting for the 7 surrogate variables were z-score normalized prior to Ward hierarchal clustering.

FIGS. 3A-3E illustrate OXPHOS up-regulation in premalignant lesion biopsies. FIG. 3A shows the mean baseline OCR/ECAR ratio measured in human bronchial biopsies cultures from PMLs (pink, n=6) was 2.5 fold higher than the biopsies of normal airway epithelium (gray n=6) (p=0.035). Error bars represent standard error of the mean. FIG. 3B shows bioenergetic studies testing mitochondrial function demonstrate PMLs (pink) have a significantly (˜1.5 fold) higher maximal respiration (p=0.022). Error bars represent standard error of the mean. FIG. 3C and FIG. 3D show mitochondrial enumeration by FACS analysis of MitoTraker GFP suggests increased OCR is not reliant on increase mitochondria as the difference in GFP per cell was not significant (p=0.150). FIG. 3E shows representative images of TOMM22 and COX IV staining in which expression of both proteins is increased in low and moderate dysplastic lesions in both human and NTCU-mouse PMLs. (Magnification 400×).

FIGS. 4A-4C shows that PML-associated gene expression alterations in the field are concordant with SCC-related datasets. The genes up-regulated in the field of subjects with PMLs are red and genes down regulated in blue. GSEA identified the significant enrichment of the lung cancer-related gene expression signatures shown in this ranked list. The black vertical lines represent the position of the genes in the gene set in the ranked list and the height corresponds to the magnitude of the running enrichment score from GSEA. FIG. 4A shows top differentially expressed genes from analysis of TCGA RNA-Seq data comparing lung SCC and matched adjacent normal tumor tissue. FIG. 4B shows Ooi et al. gene sets for early gene expression changes defined by genes altered between premalignant and normal tissue and between tumor and normal tissue (p<0.05) using laser capture microdissected (LCM) epithelium from the margins of resected SCC tumors. FIG. 4C shows top differentially expressed genes from analysis of cytologically normal bronchial epithelial cells from smokers with and without lung cancer (GSE4115).

FIGS. 5A-5B show performance of an airway biomarker in detecting the presence and progression of premalignant lesions. The ROC curves demonstrate the biomarker performance. FIG. 5A is a ROC curve (AUC=0.92) showing biomarker performance based on predictions of the presence of PMLs in the validation samples (n=17), red line. Shuffling of class labels (n=100 permutations) produced an average ROC curve (black line) with a significantly lower AUC (p<<0.001). FIG. 5B is a ROC curve (AUC=0.75) showing biomarker performance based on changes in biomarker score over time in detecting PML regression or stable/progression.

FIG. 6 shows unsupervised hierarchal clustering of genes associated with smoking status. The weighted voting algorithm was trained on z-score normalized microarray data (GSE7895) across 94 genes differentially expressed between current and never smokers and used to predict smoking status in log 2-transformed counts per million (cpm) that were z-score normalized from the 82 mRNA-Seq samples. The heatmap shows the results of unsupervised Ward hierarchal clustering across the 82 mRNA-Seq samples and the 94 genes. The row color label indicates if genes were up-regulated (red) or down-regulated (green) in current smokers compared to never smokers in GSE7895. The lower column color labels indicate the smoking status in the clinical annotation (self-report) with light gray indicating former smokers and dark gray indicating current smokers. The upper column color labels indicate the predicted class of the samples based on the 94 genes with white indicating former smokers and black indicating current smokers. Log 2-cpm mRNA-Seq data was z-score normalized prior to clustering.

FIGS. 7A-7H show cellular metabolism in cancer cell lines and in the airway field associated with premalignant lesions FIG. 7A shows GSVA scores were calculated based on genes in KEGG OXPHOS pathway and KEGG, Biocarta, and Reactome Glycolysis pathways in the CCLE cell lines highlighting the H1229 (green) (high OXPHOS and moderate glycolysis), SW900 (red) (moderate OXPHOS and low glycolysis) and H2805 (blue) ((low OXPHOS and moderate glycolysis). FIG. 7B shows baseline OCR/ECAR ratio values for the cancer cells lines demonstrating the relationship between elevated OXPHOS GSVA scores and oxygen consumption. FIG. 7C shows elevation of respiratory capacity associated with high OXPHOS gene score in response to mitochondrial perturbation. FIG. 7D shows elevated ECAR response in the H1299 and H205 is associated with the moderate glycolysis GSVA score, however, although the SW900 glycolysis GSVA scores agree with baseline ECAR, in the state of repressed OXPHOS, glycolysis is activated. FIG. 7E shows enumeration of mitochondria within each cancer cell suggests that increased GSVA scores for OXPHOS or glycolysis did not correlate with mitochondrial number. H2085 cells had the lowest OXPHOS GSVA score, the lowest basal OCR, and the lowest respiratory capacity, but their mitochondrial content was significantly greater that H1299 and SW900 (p=0.03). FIG. 7F shows cell area (FSC-A) is correlated with mitochondrial number (fluorescence of MitoTracker Green FM). FIG. 7G shows GSVA scores were calculated based on genes in KEGG OXPHOS pathway. The GSVA scores for OXPHOS activity were significantly elevated in the airway field of subjects with PMLs compared to subjects without PMLs (p<0.01). FIG. 7H shows GSVA scores were calculated based on genes in the KEGG, Biocarta, and Reactome Glycolysis pathways. The mean GSVA scores were moderately elevated in the airway field of subjects with PMLs compared to subjects without PMLs.

FIG. 8 shows a biomarker discovery flowchart. Samples (n=75) were split into a discovery set (n=58) and a validation set (n=17). The pipeline was run 500 times, and each time the discovery set was randomly split into training (80% of samples, n=46) and test (20% of samples, n=12) sets. The training set samples were used to train the biomarker using all combinations of pipeline parameters, including: 1. Up-/down-regulation ratio: TRUE or FALSE; 2. Data type: raw counts, RPKM or CPM; 3. Gene filter: genes with signal in at least 1%, 5%, 10%, or 15% of samples; 4. Feature selection: edgeR, edgeR correcting for gb-ratio, limma, limma correcting for gb-ratio, glmnet, random forest, DESeq, SVA, or partial AUC; 5. Gene number: 10, 20, 40, 60, 80, 100, or 200 genes (see Biomarker size); and 6. Prediction method: weighted voting, random forest, SVM, naïve bayes, or glmnet.

FIG. 9 shows that biomarker predicts dysplasia status in bronchial biopsies. ROC curve demonstrates the performance of the biomarker in distinguishing between premalignant lesion biopsies (severe=8, moderate=25, and mild dysplasia=14) and biopsies with normal histology (normal=24 and hyperplasia=20). Biomarker achieved AUC of 72% (with a 62%-83% confidence interval), sensitivity of 81% (38 of 47 dysplastic biopsies predicted correctly), and specificity of 66% (29 of 44 normal biopsies predicted correctly).

DETAILED DESCRIPTION OF THE INVENTION

Lung cancer develops in a sequenced manner Patches of lung cells gain the ability to multiply faster than their neighboring normal cells by acquiring mutations and these patches of cells are called “premalignant lesions” or “PMLs.” Some of these PMLs may progress to lung cancer. The inventions disclosed herein are based upon a biomarker that is capable of identifying and distinguishing epithelial cells from a person with lung cancer from normal epithelial cells. In particular, the inventions disclosed herein are based on the findings that exposure to carcinogens such as cigarette smoke induces smoking-related mRNA and microRNA expression alterations in the cytologically normal epithelium that lines the respiratory tract, creating an airway field of injury (1-8). Such gene expression alterations that were observed in the airway field of injury were used to develop a diagnostic test to facilitate early lung cancer lung cancer detection (9-12). Examination of gene signatures for p63 and the phosphatidylinositol 3-kinase (PI3K) pathway, revealed increased PI3K activation in the airway field of smokers with lung cancer or bronchial premalignant lesions (PMLs) (13). These results suggest the airway field of injury reflects processes associated with a precancerous disease state; however, the molecular changes have not been adequately characterized.

This is an important shortcoming because bronchial PMLs are precursors of squamous cell lung carcinoma, yet effective tools to identify smokers with PMLs at highest risk of progression to invasive cancer are lacking. Several studies report loss of heterozygosity, chromosomal aneusomy, and aberrant methylation and protein expression in bronchial PMLs (14-23). These molecular events can give rise to histological changes that can be reproducibly graded by a pathologist prior to the development of invasive carcinoma. Autofluorescence bronchoscopy can be used to detect and sample PMLs, which have a prevalence of approximately 9% for moderate dysplasia and 0.8% for carcinoma in situ (CIS) (24-26). The presence of high grade PMLs (severe dysplasia or CIS) is a marker of increased lung cancer risk in both the central and peripheral airways indicating the presence of changes throughout the airway field (27, 28).

The molecular characterization of the airway field of injury in smokers with PMLs disclosed herein provides novel insights into the earliest stages of lung carcinogenesis and identifies relatively accessible biomarkers to guide early lung cancer detection and early intervention. Accordingly, disclosed herein are novel biomarkers and gene expression signatures and related assays and methods that are able to provide information about the precancerous disease state and if this pre-cancerous disease state is progressing and/or regressing. Such biomarkers and the related assays and methods are useful for monitoring the progression of premalignant or pre-cancerous conditions in a subject by obtaining (e.g., non-invasively obtaining) a biological sample of epithelial cells from the respiratory tract of the subject (e.g., bronchial or nasal epithelial cells). In certain aspects, alterations in gene expression observed in epithelial cells that are distal to the lung tissues (e.g., nasal or buccal epithelial cells) are concordant with changes in the bronchial epithelium.

The present inventions represent a significant advance in the detection and monitoring of individuals with premalignant lesions (PMLs), particularly in comparison to the standard of care auto-fluorescence bronchoscopy techniques which are less sensitive. In addition to detecting and monitoring of PMLs, the present inventions provide means of advancing the identification of chemoprevention agents, which historically has been bounded by the difficulty of discerning premalignant phenotypes through currently available means. The present inventions further provide means of using gene expression profiling as a surrogate end point that complements both histological and marker end points used today, such as Ki67.

The biomarkers and related methods and assays disclosed herein are based in part upon the finding of a strong correlation between PMLs and the alterations in gene expression in tissues that are physically distant from the site of disease (e.g., the nasal epithelium). It has further been found that these biomarkers strongly predict whether a suspected PML is pre-malignant. The biomarkers, assays and methods disclosed herein are characterized by the accuracy with which they can detect and monitor lung cancer and their non-invasive or minimally-invasive nature. In some aspects, the assays and methods disclosed herein are based on detecting differential expression of one or more genes in airway epithelial cells and such assays and methods are based on the discovery that such differential expression in airway epithelial cells are useful for identifying and monitoring PMLs in the distant lung tissue. Accordingly, the inventions disclosed herein provide a substantially less invasive method for diagnosis, prognosis and monitoring of lung cancer using gene expression analysis of biological samples comprising airway epithelial cells.

In contrast to conventional invasive methods, such as auto-fluorescence bronchoscopy, the assays and methods disclosed herein rely on expression of certain genes in a biological sample obtained from a subject. As the phrase is used herein, “biological sample” means any sample taken or derived from a subject comprising one or more airway epithelial cells. As used herein, the phrase “obtaining a biological sample” refers to any process for directly or indirectly acquiring a biological sample from a subject. For example, a biological sample may be obtained (e.g., at a point-of-care facility, a physician's office, a hospital) by procuring a tissue or fluid sample from a subject. Alternatively, a biological sample may be obtained by receiving the sample (e.g., at a laboratory facility) from one or more persons who procured the sample directly from the subject.

Such biological samples comprising airway epithelial cells may be obtained from a subject (e.g., a subject suspected of having one or more PMLs or that is otherwise at risk for developing lung cancer) using a brush or a swab. The biological sample comprising airway epithelial cells may be collected by any means known to one skilled in the art and, in certain embodiments, is obtained in a non-invasive or minimally-invasive manner. For example, in certain embodiments, a biological sample comprising airway epithelial cells (e.g., nasal epithelial cells) may be collected from a subject by nasal brushing. Similarly, nasal epithelial cells may be collected by brushing the inferior turbinate and/or the adjacent lateral nasal wall. For example, following local anesthesia with 2% lidocaine solution, a CYROBRUSH® (MedScand Medical, Malmoδ, Sweden) or a similar device, is inserted into the nare of the subject, for example the right nare, and under the inferior turbinate using a nasal speculum for visualization. The brush is turned (e.g., turned 1, 2, 3, 4, 5 times or more) to collect the nasal epithelial cells, which may then be subjected to analysis in accordance with the assays and methods disclosed herein.

In some embodiments, methods and assays disclosed herein may be assessed or performed on a biological sample that is obtained from a subject at a suspected site of a PML (e.g., premalignant bronchial lesion). In some embodiments, the suspected site is identified as having abnormal fluorescent during auto-fluorescence bronchoscopy, although the method of identifying the suspected site is not limited. In some embodiments, the methods and assays disclosed herein may be performed on a biopsy of a suspected PML as an alternative to, or in addition to, a histological examination of the biopsy.

In certain embodiments, the biological sample does not include or comprise bronchial airway epithelial cells. For example, in certain embodiments, the biological sample does not include epithelial cells from the mainstem bronchus. In certain aspects, the biological sample does not include cells or tissue collected from bronchoscopy. In some embodiments, the biological sample does not include cells or tissue isolated from a pulmonary lesion. In some embodiments, the biological sample does not include cells or tissue isolated from a PML.

To isolate nucleic acids from the biological sample, the airway epithelial cells can be placed immediately into a solution that prevents nucleic acids from degradation. For example, if the nasal epithelial cells are collected using the CYTOBRUSH, and one wishes to isolate RNA, the brush is placed immediately into an RNA stabilizer solution, such as RNALATER®, AMBION®, Inc. One can also isolate DNA. After brushing, the device can be placed in a buffer, such as phosphate buffered saline (PBS) for DNA isolation.

The nucleic acids (e.g., mRNA) are then subjected to gene expression analysis. Preferably, the nucleic acids are isolated and purified. However, if techniques such as microfluidic devices are used, cells may be placed into such device as whole cells without substantial purification. In one embodiment, airway epithelial cell gene expression is analyzed using gene/transcript groups and methods of using the expression profile of these gene/transcript groups in diagnosis and prognosis of lung diseases. In some embodiments, differential expression of the one or more genes determined with reference to the one or more of the 280 genes set forth in Table 3.

As used herein, the term “differential expression” refers to any qualitative or quantitative differences in the expression of the gene or differences in the expressed gene product (e.g., mRNA or microRNA) in the airway epithelial cells of the subject. A differentially expressed gene may qualitatively have its expression altered, including an activation or inactivation, in, for example, the presence of absence of cancer and, by comparing such expression in airway epithelial cell to the expression in a control sample in accordance with the methods and assays disclosed herein, and the presence or absence of PMLs may be determined and their progression or regression monitored.

In certain embodiments, the methods and assays disclosed herein are characterized as being much less invasive relative to, for example, bronchoscopy. The methods provided herein not only significantly increase the sensitivity or diagnostic accuracy of detecting and monitoring PMLs, but in certain aspects also make the analysis faster, much less invasive and thus much easier for the clinician to perform. In some embodiments, the likelihood that the subject has a PML or the likelihood that such a PML will progress to lung cancer is also determined based on the presence or absence of one or more secondary factors or diagnostic indicia of lung cancer, such as the subject's smoking history or status, or the results of previously performed imaging studies (e.g., chest CT scans). When the biomarkers, assays and methods of the present invention are combined with, for example, one or more relevant secondary factors (e.g., a subject's smoking history), the sensitivity and accuracy of detecting PMLs or their progression to lung cancer may be dramatically enhanced, enabling the detection of PMLs or their progression to lung cancer at an earlier stage, and by providing far fewer false negatives and/or false positives. As used herein, the phrase “secondary factors” refers broadly to any diagnostic indicia that would be relevant for determining a subject's risk of having or developing lung cancer. Exemplary secondary factors that may be used in combination with the methods or assays disclosed herein include, for example, imaging studies (e.g., chest X-ray, CT scan, etc.), the subject's smoking status or smoking history, the subject's family history and/or the subject's age. In certain aspects, when such secondary factors are combined with the methods and assays disclosed herein, the sensitivity, accuracy and/or predictive power of such methods and assays may be further enhanced. In some aspects, the methods and assays described herein are performed on a patient with a positive result in an imaging study (e.g., chest X-ray, CT scan, etc.). In some aspects, the methods or assays disclosed herein are used to confirm or rule out a positive result in an imaging study (e.g., chest X-ray, CT scan, etc.). In some aspects, the methods or assays disclosed herein are used to determine whether a positive result in an imaging study warrants a further invasive procedure (e.g., bronchoscopy), chemoprophylaxis, and/or chemotherapy.

The present inventors have discovered that PMLs and normal lung cells use different pathways to produce energy and survive and have harnessed this difference to develop the biomarker and related assays and methods disclosed herein. In some embodiments, the biological sample comprising the subject's airway epithelial cells (e.g., nasal or buccal epithelial cells) are analyzed for the expression of certain genes or gene transcripts corresponding to such metabolic pathways, either individually or in groups or subsets. In one embodiment, the inventions disclosed herein provide a group of genes corresponding to one or more pathways (e.g., metabolic pathways) that are significantly enriched in genes that are up- or down-regulated in the presence of PMLs (e.g., one or more pathways identified in Dataset 2) and that may be analyzed to determine the presence or absence of PMLs and/or their progression to lung cancer (e.g., adenocarcinoma, squamous cell carcinoma, small cell cancer and/or non-small cell cancer) from a biological sample comprising the subject's airway epithelial cells. For example, in certain aspects the biological sample may be analyzed to determine the differential expression of one or more genes from pathways involved in oxidative phosphorylation (OXPHOS), the electron transport chain (ETC), and mitochondrial protein transport to determine whether the subject has PMLs or is at risk of developing lung cancer. Other up-regulated pathways included DNA repair and the HIF1A pathway. Down-regulated pathways included the STATS pathway, the JAK/STAT pathway, IL-4 signaling, RAC1 regulatory pathway, NCAM1 interactions, collagen formation, and extracellular matrix organization.

In certain embodiments, the airway epithelial cells are analyzed using at least one and no more than 280 of the genes listed in Table 3. For example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 10-15, about 15-20, about 20-30, about 30-40, about 40-50, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, 210, 220, 230, 240, 250, 260, 270 or 275 or a maximum of the 280 genes as listed on Table 3.

Examples of the gene transcript groups useful in the diagnostic and prognostic assays and methods of the invention are set forth in Table 3. The present inventors have determined that taking any group that has at least about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275 or more of the Table 3 genes provides a much greater PML detection sensitivity than chance alone. Preferably one would analyze the airway epithelial cells using more than about 20 of these genes, for example about 20-280 and any combination between, for example, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and so on. In some instances, the present inventors have determined that one can enhance the sensitivity or diagnostic accuracy of the methods and assays disclosed herein by adding additional genes to any of these specific groups. For example, in certain aspects, the accuracy of such methods may approach about 70%, about 75%, about 80%, about 82.5%, about 85%, about 87.5%, about 88%, about 90%, about 92.5%, about 95%, about 97.5%, about 98%, about 99% or more by evaluating the differential expression of more genes from the set (e.g., the set of genes set forth in Table 3).

In some embodiments, the presence of PMLs or their progression/regression is made by comparing the expression of the genes or groups of genes set forth in, for example Table 3, by the subject's airway epithelial cells to a control subject or a control group (e.g., a positive control with confirmed PMLs or a confirmed diagnosis of lung cancer). In certain embodiments, an appropriate control is an expression level (or range of expression levels) of a particular gene that is indicative of the known presence of PMLs or a known lung cancer status. An appropriate reference can be determined experimentally by a practitioner of the methods disclosed herein or may be a pre-existing expression value or range of values. When an appropriate control is indicative of lung cancer, a lack of a detectable difference (e.g., lack of a statistically significant difference) between an expression level determined from a subject in need of characterization or diagnosis of lung cancer and the appropriate control may be indicative of lung cancer in the subject. When an appropriate control is indicative of the presence of PMLs or lung cancer, a difference between an expression level determined from a subject in need of characterization or determination of PMLs or diagnosis of lung cancer and the appropriate reference may be indicative of the subject being free of PMLs or lung cancer.

Alternatively, an appropriate control may be an expression level (or range of expression levels) of one or more genes that is indicative of a subject being free of PMLs or lung cancer. For example, an appropriate control may be representative of the expression level of a particular set of genes in a reference (control) biological sample obtained from a subject who is known to be free of PMLs or lung cancer. When an appropriate control is indicative of a subject being free of PMLs or lung cancer, a difference between an expression level determined from a subject in need of detection of PMLs or the diagnosis of lung cancer and the appropriate reference may be indicative of the presence of PMLs and/or lung cancer in the subject. Alternatively, when an appropriate reference is indicative of the subject being free of PMLs or lung cancer, a lack of a detectable difference (e.g., lack of a statistically significant difference) between an expression level determined from a subject in need of detection of PMLs or diagnosis of lung cancer and the appropriate reference level may be indicative of the subject being free of PMLs and/or lung cancer.

The control groups can be or comprise one or more subjects with a confirmed presence of PMLs, positive lung cancer diagnosis, a confirmed absence of PMLs or a negative lung cancer diagnosis. Preferably, the genes or their expression products in the airway epithelial cell sample of the subject are compared relative to a similar group, except that the members of the control groups may not have PMLs and/or lung cancer. For example, such a comparison may be performed in the airway epithelial cell sample from a smoker relative to a control group of smokers who do not have PMLs or lung cancer. The transcripts or expression products are then compared against the control to determine whether increased expression or decreased expression can be observed, which depends upon the particular gene or groups of genes being analyzed, as set forth, for example, in Table 3. In certain embodiments, at least 50% of the gene or groups of genes subjected to expression analysis must provide the described pattern. Greater reliability is obtained as the percent approaches 100%. Thus, in one embodiment, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% of the one or more genes subjected to expression analysis demonstrate an altered expression pattern that is indicative of the presence or absence of PMLs or lung cancer, as set forth in, for example, Table 3. Similarly, in one embodiment, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% of the one or more genes involved in a pathways set forth in Dataset 2 are subjected to expression analysis and demonstrate an altered expression pattern that is indicative of the subject's cancer status.

Any combination of the genes and/or transcripts of Table 3 can be used in connection with the assays and methods disclosed herein. In one embodiment, any combination of at least 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80, 80-90, 90-100, 100-120, 120-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270 or 270-280 genes selected from the group consisting of genes or transcripts as shown in the Table 3.

The analysis of the gene expression of one or more genes may be performed using any gene expression methods known to one skilled in the art. Such methods include, but are not limited to expression analysis using nucleic acid chips (e.g. Affymetrix chips) and quantitative RT-PCR based methods using, for example real-time detection of the transcripts. Analysis of transcript levels according to the present invention can be made using total or messenger RNA or proteins encoded by the genes identified in the diagnostic gene groups of the present invention as a starting material. In certain aspects, analysis of transcript levels according to the present invention can be made using micronRNA. In the preferred embodiment the analysis is an immunohistochemical analysis with an antibody directed against proteins comprising at least about 10-20, 20-30, preferably at least 36, at least 36-50, 50, about 50-60, 60-70, 70-80, 80-90, 96, 100-180, 180-200, 200-250 or 250-280 of the proteins encoded by the genes and/or transcripts as shown in Table 3.

The methods of analyzing expression and/or determining an expression profile of the one or more genes include, for example, Northern-blot hybridization, ribonuclease protection assay, and reverse transcriptase polymerase chain reaction (RT-PCR) based methods. In certain aspects, the different RT-PCR based techniques are a suitable quantification method for diagnostic purposes of the present invention, because they are very sensitive and thus require only a small sample size which is desirable for a diagnostic test. A number of quantitative RT-PCR based methods have been described and are useful in measuring the amount of transcripts according to the present invention. These methods include RNA quantification using PCR and complementary DNA (cDNA) arrays (Shalon, et al., Genome Research 6(7):639-45, 1996; Bernard, et al., Nucleic Acids Research 24(8): 1435-42, 1996), real competitive PCR using a MALDI-TOF Mass spectrometry based approach (Ding, et al., PNAS, 100: 3059-64, 2003), solid-phase mini-sequencing technique, which is based upon a primer extension reaction (U.S. Pat. No. 6,013,431, Suomalainen, et al., Mol. Biotechnol. June; 15(2): 123-31, 2000), ion-pair high-performance liquid chromatography (Doris, et al., J. Chromatogr. A May 8; 806(1):47-60, 1998), and 5′ nuclease assay or real-time RT-PCR (Holland, et al., Proc Natl Acad Sci USA 88: 7276-7280, 1991).

The presently described gene expression profile can also be used to screen for subjects with confirmed PMLs to determine whether such subject are susceptible to or otherwise at risk for developing lung cancer. For example, a current smoker of advanced age (e.g., 70 years old) with PMLs may be at an increased risk for developing lung cancer and may represent an ideal candidate for the assays and methods disclosed herein. Moreover, the early detection of lung cancer in such a subject may improve the subject's overall survival. Accordingly, in certain aspects, the assays and methods disclosed herein are performed or otherwise comprise an analysis of the subject's secondary risk factors for developing cancer. For example, one or more secondary factors selected from the group consisting of advanced age (e.g., age greater than about 40 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years or more), smoking status, the presence of a lung nodule greater than 3 cm on CT scan and the time since the subject quit smoking. In certain embodiments, the assays and methods disclosed herein further comprise a step of considering the presence of any such secondary factors to inform the determination of whether the subject has PMLs or whether such PMLs are likely to progress to lung cancer.

As used herein, a “subject” means a human or animal Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. In certain embodiments, the subject is a mammal (e.g., a primate or a human). The subject may be an infant, a toddler, a child, a young adult, an adult or a geriatric. The subject may be a smoker, a former smoker or a non-smoker. The subject may have a personal or family history of cancer. The subject may have a cancer-free personal or family history. The subject may exhibit one or more symptoms of lung cancer or other lung disorder (e.g., emphysema, COPD). For example, the subject may have a new or persistent cough, worsening of an existing chronic cough, blood in the sputum, persistent bronchitis or repeated respiratory infections, chest pain, unexplained weight loss and/or fatigue, or breathing difficulties such as shortness of breath or wheezing. The subject may have a lesion, which may be observable by computer-aided tomography or chest X-ray. The subject may be an individual who has undergone a bronchoscopy or who has been identified as a candidate for bronchoscopy (e.g., because of the presence of a detectable lesion or suspicious imaging result). The terms, “patient” and “subject” are used interchangeably herein. In some embodiments, the subject is at risk for developing lung cancer. In some embodiments, the subject has PMLs or lung cancer and the assays and methods disclosed herein may be used to monitor the progression of the subject's disease or to monitor the efficacy of one or more treatment regimens.

In some embodiments, the methods and assays disclosed herein are useful for identifying subjects that are candidates for enrollment in a clinical trial to assess the efficacy of one or more chemotherapeutic agents. In certain aspects, the methods and assays disclosed herein are useful for determining a treatment course for a subject. For example, such methods and assays may involve determining the expression levels of one or more genes (e.g., one or more of the genes set forth in Table 3) in a biological sample obtained from the subject, and determining a treatment course for the subject based on the expression profile of such one or more genes. In some embodiments, the treatment course is determined based on a risk-score derived from the expression levels of the one or more genes analyzed. The subject may be identified as a candidate for a particular intervention or treatment based on an expression profile that indicates the subject's likelihood of having PMLs that will progress lung cancer. Similarly, the subject may be identified as a candidate for an invasive lung procedure (e.g., transthoracic needle aspiration, mediastinoscopy, lobectomy, or thoracotomy) based on an expression profile that indicates the subject has a relatively high likelihood of having PMLs or a high likelihood that such PMLs will progress to lung cancer (e.g., greater than 60%, greater than 70%, greater than 80%, greater than 90%). Conversely, the subject may be identified as not being a candidate for interventional therapy or an invasive lung procedure based on an expression profile that indicates the subject has a relatively low likelihood (e.g., less than 50%, less than 40%, less than 30%, less than 20%) of having PMLs or a low likelihood that such PMLs will progress to lung cancer. In some embodiments, a health care provider may elect to monitor the subject using the assays and methods disclosed herein and/or repeat the assays or methods at one or more later points in time, or undertake further diagnostics procedures to rule out PMLs or lung cancer. Also contemplated herein is the inclusion of one or more of the genes and/or transcripts presented in, for example, Table 3 into a composition or a system for detecting lung cancer in a subject. For example, any one or more genes and or gene transcripts from Table 3 may be added as a PML marker or lung cancer marker for a gene expression analysis. In some aspects, the present inventions relate to compositions that may be used to determine the expression profile of one or more genes from a subject's biological sample comprising airway epithelial cells. For example, compositions are provided that consist essentially of nucleic acid probes that specifically hybridize with one or more genes set forth in Table 3. These compositions may also include probes that specifically hybridize with one or more control genes and may further comprise appropriate buffers, salts or detection reagents. In certain embodiments, such probes may be fixed directly or indirectly to a solid support (e.g., a glass, plastic or silicon chip) or a bead (e.g., a magnetic bead).

The compositions described herein may be assembled into diagnostic or research kits to facilitate their use in one or more diagnostic or research applications. In some embodiments, such kits and diagnostic compositions are provided that comprise one or more probes capable of specifically hybridizing to up to 5, up to 10, up to 25, up to 50, up to 100, up to 200, up to 225, up to 250 or up to 280 genes set forth in Table 3 or their expression products (e.g., mRNA or microRNA). In some embodiments, each of the nucleic acid probes specifically hybridizes with one or more genes selected from those genes set forth in Table 3, or with a nucleic acid having a sequence complementary to such genes. A kit may include one or more containers housing one or more of the components provided in this disclosure and instructions for use. Specifically, such kits may include one or more compositions described herein, along with instructions describing the intended application and the proper use and/or disposition of these compositions. Kits may contain the components in appropriate concentrations or quantities for running various experiments.

The articles “a” and “an” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

EXAMPLES Example 1 Patient Population

Bronchial airway brushings were obtained during autofluorescence bronchoscopy procedures between June 2000 and March 2011 from subjects in the British Columbia Lung Health Study at the British Columbia Cancer Agency (BCCA) (Vancouver, BC) (29) and between December 2009 and March 2013 from subjects in the High-Risk Lung Cancer-Screening Program at Roswell Park Cancer Institute (RPCI) (Buffalo, N.Y.) (detailed cohort information in the Methods section below). Premalignant Lesions were sampled (if present) using endobronchial biopsy, graded by a team of pathologists at BCCA or RPCI, and the worst histology observed was recorded. Bronchial brushes of normal-appearing epithelium from 84 BCCA subjects (1 brush per subject) with and without PMLs were selected to undergo mRNA-Seq while ensuring balanced clinical covariates. Fifty-one bronchial brushes of normal-appearing epithelium from 23 RPCI subjects were also profiled by mRNA-Seq (18 subjects had 2 procedures, and 5 subjects had 3 procedures). The RPCI samples were utilized in biomarker validation to calculate changes in the biomarker score between sequential procedures. Sets of samples were classified as stable/progressive if the worst histological grade at the second time point for a given patient remained the same or worsened, and regressive if the worst histological grade at the second time point improved. The Institutional Review Boards (IRBs) of all participating institutions approved the study and all subjects provided written informed consent.

RNA-Seq Library Preparation, Sequencing and Data Processing

Total RNA was extracted from bronchial brushings using miRNeasy Mini Kit (Qiagen). Sequencing libraries were prepared from total RNA samples using Illumina® TruSeq® RNA Kit v2 and multiplexed in groups of four using Illumina® TruSeq® Paired-End Cluster Kit. Each sample was sequenced on the Illumina® HiSeq® 2500 to generate paired-end 100 nucleotide reads. Demultiplexing and creation of FASTQ files were performed using Illumina CASAVA v1.8.2. For the BCCA samples, reads were aligned to hg19 using TopHat v2.0.4. The insert size mean and standard deviation were determined using the alignments and MISO (32). Reads were realigned using TopHat and the insert size parameters. Alignment and quality metrics were calculated using RSeQC v2.3.3. Gene count estimates were derived using HTSeq-count v0.5.4 (33) and the Ensembl v64 GTF file. Gene filtering was conducted on normalized counts per million (cpm) calculated using R v3.0.0 and edgeR v3.4.2 using a modified version of the mixture model in the SCAN. UPC Bioconductor package (34). A gene was included in downstream analyses if the mixture model classified it as “on” (i.e. “signal”) in at least 15% of the samples. For the RPCI samples, gene counts were computed using RSEM (v1.2.1) (30) and Bowtie (v1.0.0) (31) with Ensembl 74 annotation. The data is available from NCBI's Gene Expression Omnibus (GEO) using the accession ID GSE79315.

Data Analysis for the BCCA Samples

Sample and gene filtering yielded 13,870 out of 51,979 genes and 82 samples (n=2 excluded due to quality or sex annotation mismatches) for analysis. Data from Beane et al. (3) was used to predict the smoking status of the 82 samples (Dataset 1, FIG. 6 and Methods) used in all further analysis. Airway brushings were dichotomized into two groups: samples with no evidence of PMLs (samples with no abnormal fluorescing areas or biopsies having normal or hyperplasia histology, n=25); and samples with evidence of PMLs (biopsies having mild, moderate, or severe dysplasia, n=50). Brushes with a worst histology of metaplasia (n=7) were excluded from the dichotomized groups. The limma (35), edgeR (36) and sva packages (37) were used to identify differentially expressed genes associated with presence of PMLs using normalized voom-tranformed (38) data and surrogate variable analysis using the first 7 surrogate variables (Table 51). Gene set enrichment analyses were conducted using ROAST (39) and GSEA (40), and GSVA (41). The Molecular Signatures Database (MSigDb) v4 Entrez ID Gene Sets were converted to Ensembl IDs using BioMart. Additional gene sets were created from CEL files or RNA-Seq counts from The Cancer Cell Line Compendium (CCLE), SCC tumor and adjacent normal tissue from TCGA, GSE19188, GSE18842, and GSE4115 (Supplemental Methods).

Cell Culture

The human bronchial epithelial biopsy cell cultures (Table S2) were obtained from the Colorado Lung SPORE Tissue Bank and cultured in Bronchial Epithelial Growth Media (BEGM). Human non-small cell lung cancer (NSCLC) cell lines were purchased from ATCC and short tandem repeat (STR) profiles were verified at the time of use by the Promega Gene Print® 10 system at the Dana Faber Cancer Institute. H1299, H2085 and SW900 cells were cultured in RPMI supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and H2085 cells were cultured in ALC-4 media. All cells were grown in a 37° C. humidified incubator with 5% CO₂.

Bioenergetics Studies

Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using the XF96 Extracellular Flux Analyzer instrument (Seahorse Bioscience Inc). Briefly, approximately 30,000 cancer cells/well or approximately 40,000 bronchial epithelial biopsy cells/well (higher numbers due to slow growth rate) were seeded on XF96 cell culture plates and grown overnight. Prior to running the assay, media was replaced with Seahorse base media (2 mM (milimole/L) L-glutamine) and placed at 37° C. and 0% CO₂ for approximately 30 minutes. The XF Cell Mito Stress Test kit and protocol were utilized to examine mitochondrial function. Measurements were taken every 5 minutes over 80 minutes. To modulate mitochondrial respiration, 504 oligomycin, 1 μM FCCP and 504 antimycin A were used. Prism software v6 was used to calculate t-statistics for baseline OCR comparisons and a 2-way ANOVA was conducted to compare OCR and ECAR measurements.

Mitochondrial Enumeration Using Flow Cytometry

Using an established protocol (40), cell cultures (5×10⁵ cells/10 cc dish of bronchial biopsy cultures and cancer cell cultures) were grown overnight and exposed to 120 uM MitoTracker Green FM in media free of FBS for 30 min at 37° C. humidified incubator with 5% CO₂. Cells were subsequently collected, washed in PBS and resuspended in 0.5 mL PBS-EDTA and 1 uL of propidium iodide (PI) was added to distinguish live/dead cells. MitoTracker FM and PI were measured using a BD LSRII flow cytometer and BD FACS Diva software (6.2.1). Data was analyzed using FlowJo (10.2), gating out doublets and dead cells, and normalizing mean fluorescence to the number of cell counts.

Immunohistochemistry

Formalin-fixed, paraffin-embedded (FFPE) sections of human PMLs sampled from high-risk subjects undergoing screening for lung cancer were provided by RPCI as part of an IRB-approved study detailed below (Table S3). Dr. Candace Johnson at RPCI provided the PIPE lung sections from the N-nitroso-tris-chloroethylurea (NTCU) mouse model of lung SCC, from mice treated with 25 ml of 40 mmol/L NTCU for 25 weeks in accordance with the Institutional Animal Care and Use Committee approved protocol (42). Antibody dilutions and immunohistochemistry methods were detailed in the Supplemental Methods. Briefly, slides were de-paraffinized and rehydrated. For antigen retrieval, slides were heated in citrate buffer. Slides were subsequently incubated in primary antibody (Translocase of the Outer Mitochondrial Membrane 22 (TOMM22): mouse tissue 1:300 and human 1:1,200 (Abcam), and Cytochrome C Oxidase subunit IV (COX4I1): mouse tissue 1:500 and human 1:5,000 (Abcam)) diluted in 1% Bovine Serum Albumin (BSA). Signal was amplified using an ABC kit (Vector Labs). To reveal endogenous peroxidase activity, slides were incubated in a 3,3′-Diaminobenzidine (DAB) solution. Slides were rinsed, counterstained with hematoxylin, dehydrated in graded alcohol followed by xylene and cover slipped.

Biomarker Development and Validation

A gene expression biomarker discovery pipeline was developed to test thousands of parameter combinations (6,160 predictive models) to identify a biomarker capable of distinguishing between samples from subjects with and without PMLs. Samples were first assigned by batch (sequencing lane) to either a discovery set (n=58) or a validation set (n=17), and the validation set was excluded from biomarker development (FIG. S2 and Supplemental Methods). The biomarker was developed using subsets of the discovery set established by randomly splitting the samples into training (80%, n=46) and test (20%, n=12) sets 500 times. Model performance was assessed using standard metrics for both the training and test sets (Supplemental Methods). The biomarker pipeline was also used to develop biomarkers for sex and smoking status as well as randomized class labels for all phenotypes (serving as positive and negative controls, respectively). A final model (biomarker) was selected (Supplemental Methods) and its ability to distinguish between samples with and without PMLs was tested in a validation set (n=17). In addition, using the bronchial brushings collected longitudinally from subjects at RPCI, we tested whether or not differences in biomarker scores over time were reflective of progression of PMLs (n=28 matched time point pairs) (Supplemental Methods).

Example 2 Results Subject Population

The study design used 126 bronchial brushings obtained via autofluorescence bronchoscopy at the BCCA and RPCI for differential gene expression and pathway analysis, as well as for biomarker development and validation (FIG. 1). A dataset consisting of samples collected from BCCA subjects with (n=50) and without (n=25) PMLs (n=25) was used to derive a gene expression signature associated with the presence of dysplastic PMLs. Important clinical covariates such as COPD and reported smoking history as well as alignment statistics from the mRNA-Seq data were not significantly different between the two groups (Table 1 and Table 2). For biomarker development, the 75 BCCA samples were split by batch and used in biomarker discovery (n=58) and validation (n=17) (Tables S4 and S5). The change in biomarker score as a predictor of progression of PMLs was then tested in the 51 RPCI samples (Tables S5 and S6).

Transcriptomic Alterations in the Airway Field of Injury Associated with the Presence of PMLs

The present inventors identified 280 genes significantly differentially expressed between subjects with and without PMLs (FDR<0.002, FIG. 2). Utilizing the Molecular Signatures Database v4 (MSigDB) canonical pathways, the present inventors identified 170 pathways significantly enriched in genes up- or down-regulated in the presence of PMLs using ROAST (39) (FDR<0.05, Dataset 2). Pathways involved in oxidative phosphorylation (OXPHOS), the electron transport chain (ETC), and mitochondrial protein transport were strongly enriched among genes up-regulated in the airways of subjects with PMLs. Other up-regulated pathways included DNA repair and the HIF1A pathway. Down-regulated pathways included the STATS pathway, the JAK/STAT pathway, IL4 signaling, RAC1 regulatory pathway, NCAM1 interactions, collagen formation, and extracellular matrix organization.

OXPHOS is Increased in PML Cell Cultures and Biopsies of Increasing Severity

The ETC and OXPHOS pathways, which involve genes distributed between the complexes I-IV of the ETC and ATP synthase, were highly activated in the airway field in the presence of PMLs. The present inventors wanted to determine if the functional activity of these pathways was similarly altered in PMLs compared to normal tissue. Cellular bioenergetics were conducted by measuring oxygen consumption rate (OCR) as a measure of ETC/OXPHOS and extracellular acidification rate (ECAR) as a measure of glycolysis (anerobic respiration) and MitoTraker Green FM as a measure of mitochondrial content in primary cell cultures derived from bronchial biopsies. Additionally, the present inventors performed immunohistochemistry of select OXPHOS-related genes in mouse and human dysplastic lesions and normal tissue to measure protein levels.

The present inventors established a significant concordance between ETC/OXPHOS gene expression and cellular bioenergetics in NSCLC cell lines (FIGS. 7A-7F). Next, using primary cell cultures derived from normal to severe dysplastic tissue (Table S2), the present inventors observed that the mean baseline OCR values were 2.5 fold higher in the cultures from PMLs compared to controls (p<0.001, FIG. 3A). Baseline ECAR values were also higher in PML cultures compared to controls, but to a lesser extent (1.5 fold, p<0.001), reflecting predictions based on mRNA-Seq field data (FIGS. 7G-7H). There was a greater reduction in OCR in PMLs immediately following oligomycin treatment (p<0.001) suggesting an increased dependence on OXPHOS for ATP production to meet energetic demands. In addition, the mean spare respiratory capacity following the release of the proton gradient was elevated by approximately 1.5 fold in the PML cultures compared to controls indicating increased ability to respond to energy demands (43). Lastly, treatment with antimycin A resulted in a greater reduction of OCR in PML cultures (p<0.001, FIG. 3B), suggesting that oxygen consumption in the lesions is dependent on increased ETC components in complex III. No significant changes to ECAR were detected in response to mitochondrial perturbations. Furthermore to examine if the increased OXPHOS was a result of increased mitochondrial biogenesis in PML cultures, cells were incubated with MitoTraker FM to stain for mitochondria content and fluorescence enumerated using flow cytometry revealed no significant difference between PML and controls (p=0.15, FIG. 3C-D).

Additionally, the present inventors found elevated protein levels of Translocase of the Outer Mitochondrial Membrane 22 (TOMM22) and Cytochrome C Oxidase subunit IV (COX4I1) in low/moderate grade dysplastic lesions compared to normal tissue (FIG. 3C) using tissues from human bronchial biopsy FFPE sections (Table S3) and whole lung sections from the NTCU mouse model of SCC. The results suggest that PMLs are more ETC- and OXPHOS-dependent and express OXPHOS-related proteins at higher levels compared to normal tissue.

PML-Associated Gene Expression Alterations in the Airway Field are Involved in Lung Squamous Cell Carcinogenesis

To further extend the connection between the airway field and PMLs, the present inventors examined the relationship between PML-associated genes in the airway field and other lung cancer-related datasets. The present inventors identified genes differentially expressed between lung tumor tissue (primarily squamous) and normal lung tissue in three different datasets (TCGA, GSE19188, and GSE18842). Genes associated with lung cancer in all datasets were significantly (FDR<0.05) enriched by GSEA, concordantly with gene expression changes associated with the presence of PMLs in the field (FIG. 4A and Dataset 3). Extending beyond the lung tumor, similar enrichment (FDR<0.05) was found using early, stepwise, and late gene expression changes in SCC identified by Ooi et al. (44) (FIG. 4B and Dataset 3) and among genes associated with lung cancer in the airway field of injury (GSE4115, FIG. 4C and Dataset 3). These results support the concept that early events in lung carcinogenesis can be observed throughout the respiratory tract, even in cells that appear cytologically normal.

Development and Validation of a Biomarker for PML Detection and Monitoring

The airway brushings from BCCA subjects with and without PMLs were leveraged to build a biomarker predictive of the presence of PMLs. The biomarker consisted of 200 genes (of which 91 overlapped with the gene signature in FIG. 2) and achieved a ROC-curve AUC of 0.92, sensitivity of 0.75 (9/12 samples with PMLs predicted correctly), and specificity of 1.00 (5/5 samples without PMLs predicted correctly) in independent validation samples (n=17, FIG. 5A). In addition, the biomarker was used to score an independent set of longitudinally collected bronchial brushings from RPCI subjects (FIG. 1). Biomarker scores were calculated for each sample, and the difference in biomarker scores between sequential procedures (n=28 time point pairs, Supplemental Methods) was predictive of whether the worst PML histology observed during the baseline procedure regressed or whether it was stable or progressed with an AUC of 0.75 (FIG. 5B).

Biomarker Predicts Dysplasia Status in Bronchial Biopsies

Abnormal fluorescing areas were biopsied during auto-fluorescence bronchoscopy of 91 subjects. Biopsies from 47 of the subjects were determined to be premalignant legions (severe, moderate or mild dysplasia) via histology. Biopsies from 44 of the subjects were determined to be normal (normal or hyperplasia) via histology. The ability of the biomarker to predict dysplasia status was assessed. FIG. 9 shows an ROC curve demonstrating the performance of the biomarker in distinguishing between premalignant lesion biopsies (severe=8, moderate=25, and mild dysplasia=14) and biopsies with normal histology (normal=24 and hyperplasia=20). Biomarker achieved AUC of 72% (with a 62%-83% confidence interval), sensitivity of 81% (38 of 47 dysplastic biopsies predicted correctly), and specificity of 66% (29 of 44 normal biopsies predicted correctly).

Discussion

In the foregoing studies, the present inventors identified a PML-associated gene expression signature in cytologically normal bronchial brushings and characterized the biological pathways that are dysregulated in the airway field of injury. The present inventors established that the PML-associated airway field harbors alterations observed in PMLs and in SCC. This evidence motivated the development of a biomarker that reflects the presence of PMLs and their outcome over time. The findings presented herein provide novel insights into the earliest molecular events associated with lung carcinogenesis and have the potential to impact lung cancer prevention by providing novel targets (e.g., OXPHOS) and potential biomarkers for risk stratification and monitoring the efficacy of chemoprevention agents.

The first major finding of the foregoing studies was the identification of a PML-associated field of injury. The most significantly enriched pathways among up-regulated genes in subjects with PMLs were OXPHOS, ETC, and mitochondrial protein transport. These pathways efficiently generate energy in the form of ATP by utilizing the ETC in the mitochondria. During cancer development, energy metabolism alterations are described as an increase in glycolysis and suppression of OXPHOS, known as the Warburg effect (45); however, recent studies demonstrate that OXPHOS is maintained in many tumors and can be important for progression (46). The present inventors wanted to assay for OXPHOS activation in PMLs as it may support PML progression by generating reactive oxygen species (ROS) that can induce oxidative stress, increase DNA damage, and HIF-1α pathway activation (pathways observed in our analysis).

The present inventors observed increases in both the basal OCR and the spare respiratory capacity in the PML biopsies, suggesting that PML-derived cell cultures are more ETC and OXPHOS dependent that the non-PML cultures. The present inventors also demonstrated increases in the presence of mitochondria and ETC activity marked by positive TOMM22 and COX IV staining associated with increasing PML histological grade. Several members of the mitochondrial protein import machinery (46) were significantly up-regulated (FDR<0.05) in airways with PMLs including members of the TOM complex (TOMM22, TOMM7, and TOMM20) and TIM23 complex (TIMM23, TIMM21, and TIMM17A). We observed positive staining of TOMM22 with increasing PML grade, suggesting that increased import of precursor proteins from the endoplasmic reticulum may be required to meet the energy demands of PMLs. Measurements of mitochondrial content indicated no significant differences between the normal and PML-derived cultures, and transcriptional levels of PPARGC1A, associated with mitochondrial biogenesis, were not different between subjects with and without PML indicating that increases in OXPHOS are likely independent of mitochondrial number (47-49). Increases in OXPHOS have been demonstrated to be associated with PML progression in Barret's esophagus and esophageal dysplasia (47), cervical dysplasia (48), and the dysplastic lesions that precede oral SCC (49). Collectively, these data suggest that the OXPHOS pathway may be a target for early intervention. Pre-clinical studies in the NTCU mouse model of lung SCC demonstrate the potential for targeting mitochondrial respiration by using the natural product honokiol to inhibit tumor development (50). Further investigations into the role of cellular energy metabolism in the development and progression of PMLs are needed to fully understand how to best target it for intervention in lung cancer.

Additionally, the present inventors extended the connection between the PML-associated airway field and PMLs beyond the OXPHOS pathway to processes associated with squamous cell lung carcinogenesis. By examining gene sets from multiple external studies representative of lung cancer-related processes occurring in the tumor, adjacent to the tumor, and in the upper airway, significant concordant relationships were found between the PML-associated field and processes associated with SCC tumors. Genes are similarly altered in these varied cancer-associated contexts and thus tissues in the field both adjacent to and far away from the tumor may reflect basic processes and mechanisms of lung carcinogenesis such as DNA damage as hypothesized earlier.

These observations motivated the present inventors to pursue the most translational aspect of this study, a biomarker that can detect PMLs and monitor their progression over time. The 200-gene biomarker, measured in the cytologically normal bronchial airway, achieved high performance detecting the presence of PMLs in a small test set (AUC=0.92). This biomarker may increase the sensitivity of bronchoscopy in detecting the presence of PMLs (which can be difficult to observe under white light), and thus improve identification of high-risk smokers that should be targeted for aggressive lung cancer screening programs. Additionally, the biomarker may offer wider clinical utility in early intervention trials by serving as an intermediate endpoint of efficacy (beyond Ki-67 staining for proliferation, and changes in biopsy histology). Towards this goal, the present inventors demonstrated that the change in biomarker scores over time reflected contemporaneous regressive or progressive/stable disease (AUC=0.75). This result suggests that the airway field of injury in the presence of PMLs is dynamic and that capturing the gene expression longitudinally may allow for further stratification of high-risk subjects. The potential clinical utility of the biomarker is further supported by recent work demonstrating a significant association between the development of incident lung squamous cell carcinoma and the frequency of sites that persist or progress to high-grade dysplasia (24).

Further development and testing in a larger cohort is needed to confirm the biomarker's performance, utility, and ability to predict future PML progression or regression. Additionally, longitudinal and spatial sampling would provide a greater understanding of the dynamic relationship between the normal epithelium and the PMLs as they regress or progress to SCC. Longitudinal studies would allow for more accurate characterization of the time intervals needed to observe gene expression dynamics both in the PMLs and in the airway field of injury. Spatial sampling throughout the respiratory tract, including the more accessible nasal airway that shares the tobacco-related injury with the bronchial airways (51), would allow for evaluation of the impact of distance between the PMLs and the brushing site, the range of PML histologies, and the multiplicity of PMLs that can be present simultaneously in a patient and influence the PML-associated airway field.

Despite these challenges and opportunities for future work, the present inventors have comprehensively profiled gene expression changes in airway epithelial cells in the presence of PMLs that suggest great clinical utility. Moving therapeutics and detection strategies towards an earlier stage in the disease process via molecular characterization of premalignant disease holds great promise (52, 53), and this study represents an important step towards a precision medicine approach to lung cancer prevention.

Materials and Methods

Software versions referenced

Data Processing Illumina CASAVA v1.8.2 TopHat v2.0.4 RSeQC v2.3.3 HTSeq-count v0.5.4 R v3.0.0

edgeR v3.4.2

RSEM v1.2.1 Bowtie v1.0.0 Data Analysis Limma v3.18.13

edgeR v3.4.2 sva v3.6.0

GSVA v1.10.3 Gene Expression-Based Prediction of Smoking Status

Microarray data from Beane et al. (3) Gene Expression Omnibus [GEO] (54) Accession Number GSE7895) was re-analyzed using using Robust Multi-array Average (RMA) (54) and the Ensembl CDF file v16.0.0 file website (brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/16.0.0/ensg.asp). The R package (35) was used to identify genes differentially expressed between current (n=52) and never (n=21) smokers, using the linear model presented in the paper additionally correcting for quality covariates (NUSE and RLE). Ninety-four genes (FDR<0.001) were differentially expressed between current and never smokers. The weighted voting algorithm (55) was trained on z-score normalized microarray data (n=73) across the 94 genes and used to predict smoking status in z-scored log 2-transformed counts per million (cpm) from the 82 mRNA-Seq samples.

Processing of Publically Available Datasets

Cancer Cell Line Compendium (CCLE). The Entrez ID gene expression file labeled 10/18/2012 and the sample information file were downloaded from CCLE website (broadinstitute.org/ccle/home). After matching the sample annotation to the expression file, we used ComBat (56) to adjust the data for batch effects (n=14 batches across 1019 samples). After batch correction, the lung cell lines (n=186) were selected and GSVA was used to calculate a pathway enrichment score for each lung cell line for the following pathways: KEGG oxidative phosphorylation, KEGG glycolysis gluconeogenesis, BioCarta glycolysis, and Reactome glycolysis. The GSVA scores for the glycolysis pathways were averaged per sample.

The Cancer Genome Atlas (TCGA). RSEM gene-level (Entrez IDs) counts derived from RNA-Seq data were downloaded from the TCGA data portal on Aug. 27, 2013, for lung squamous cell carcinomas and adjacent matched control tissue (n=100 samples from n=50 subjects). After applying the mixture model referenced in the paper, 14,178 out of 20,531 genes were expressed as signal in at least 15% of samples (n=15). Differential gene expression between tumor and adjacent normal tissue was determined using limma and voom-transformed data (38) via a linear model with cancer status as the main effect and a random patient effect modeled using the duplicateCorrelation function. Gene sets containing the top 200 up- and down-regulated differentially expressed genes associated with cancer status were used as input for GSEA.

Microarray Data. CEL files for GSE19188 and GSE18842 were downloaded from GEO and processed using Robust Multi-array Average (RMA) (54) and the Ensembl Gene CDF v16.0.0 file website (brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/16.0.0/ensg.asp). Samples with a median RLE greater than 0.1 or a median NUSE greater than 1.05 were excluded, yielding n=146 samples for GSE19188 and n=82 samples for GSE18842. For GSE19188, differential gene expression between squamous cell tumors (n=23) and normal lung tissue (n=64) was conducted using limma and a linear model that included RLE and NUSE covariates. For GSE18842, paired normal and tumor tissue from the same subjects (n=37 subjects, n=74 samples) were selected, and differential gene expression was conducted in an analogous manner as described above for TCGA, additionally correcting for RLE and NUSE metrics.

CEL files for GSE4115 were processed using RMA and the CDF file above. The n=164 samples described in Spira et al. (9), were used to determine genes differentially expressed in airway brushings from subjects with and without lung cancer, using limma and a linear model with terms for cancer status, RLE, NUSE, smoking status, and pack-years. Gene sets containing the top 200 up- and down-regulated differentially expressed genes associated with cancer status were used as input for GSEA.

Immunohistochemistry

Slides were de-paraffinized, rehydrated, and heated in citrate buffer for antigen retrieval. Slides were treated with 3% H₂O₂ (in methanol) to block endogenous peroxidases, incubated in 10% normal goat serum, and primary antibody (TOMM22: mouse tissue 1:300 and human 1:1,200 (Abcam), and COX IV: mouse tissue 1:500 and human 1:5,000 (Abcam)) diluted in 1% BSA. Signal was amplified using an ABC kit (Vector Labs). Slides were next incubated in a 3,3′-Diaminobenzidine (DAB) solution to reveal endogenous peroxidase activity, rinsed, counterstained with hematoxylin, dehydrated in graded alcohol followed by xylene, and cover slipped.

Biomarker Development

Upstream gene filtering. In order to provide cross-platform compatibility, the present inventors ran the biomarker discovery and validation pipelines using 11,926 genes commonly present on the RNA-Seq platform (Illumina HiSeq 2500 used with Ensembl v64 GTF) and two microarray platforms (Affymetrix GeneChip Human Gene 1.0 ST Array used with custom ENSG Homo sapiens CDF from Brainarray v11 and Affymetrix Human Genome U133A Array used with custom ENSG Homo sapiens CDF from Brainarray v16).

Data generation and summarization. Samples (n=75) were run across 4 flow cells (4 batches), and samples run in batches 1, 2, and 3 (n=58) were assigned to a discovery set, while the remaining samples (n=17) were used as an independent validation set and not included in the biomarker development. Alignments and gene level summarization were conducted as described in the paper methods. Alignment and quality metrics were calculated using RSeQC (v2.3.3) (57). Using the gene body measure computed by RSeQC, a ratio between the average read coverage at 80% of the gene length and the average coverage at 20% of the gene length was derived as an additional quality metric (gb-ratio) to assess 3′ bias per sample. The metric was highly correlated with a surrogate variable applied in the identification of differentially expressed genes, and was used as a quality control metric in the biomarker pipeline.

Biomarker discovery pipeline. The biomarker discovery pipeline has been outlined generally above. A graphical representation of data flow as well as processing and analysis steps is provided in FIG. 8. Each computational step outlined is detailed in the following sections.

Balancing signature. The present inventors tested gene signatures consisting either of an equal or unequal number of genes up- and down-regulated in subjects with dysplastic lesions.

Input data preprocessing. The present inventors tested 3 input data types. HTSeq-count (v0.5.4) (33) was used to derive gene count estimates (raw counts). In addition, Cufflinks (v2.0.2) (58) was used to derive reads per kilobase per million mapped reads (RPKM) using BAM files containing only properly paired reads. The present inventors also calculated log 2-transformed counts per million (CPM) by applying edgeR (v3.8.6) (36) to raw counts using the “TMM” method (weighted trimmed mean of M-values (59)).

Gene filtering. Signal-based gene filtering was conducted as described in detail above (Methods). In short, a gene was included in downstream analyses if the mixture model classified it as “on” in at least 1%, 5%, 10% or 15% of the samples. For CPM input data type, the present inventors recalculated CPM values using raw counts after filtering out genes.

Feature selection. To identify genes differentially expressed (DE) between samples with and without premalignant lesions (PMLs), the present inventors applied several algorithms to our filtered dataset. The algorithms used were as follows:

(1) edgeR: The present inventors applied the edgeR package (v3.8.6) (46) to raw counts only. After calculating normalization factors (calcNormFactors) and estimating common (estimateGLMCommonDisp) and tagwise (estimateGLMTagwiseDisp) dispersion factors, we identified DE genes associated with the presence of PMLs using a generalized linear model, correcting for sex, COPD status, and smoking status covariates. For balanced signatures, the sign of the log 2-fold change of expression between conditions determined gene directionality. For all models regardless of balancing, gene importance was defined by FDR-adjusted p-value from likelihood ratio tests (glmLRT).

(2) edgeRgb: The present inventors used the edgeR package as described in #1, additionally correcting for gb-ratio (described above in the Data generation and summarization section).

(3) lm: The present inventors applied the limma package (v3.22.7) (35) to CPMs, RPKMs, or voom-transformed raw counts (38). Voom transformation was applied using a linear model, adjusting for sex, COPD status, and smoking status covariates, after calculating normalization factors. The same model was used to identify DE genes associated with the presence of PMLs. For balanced signatures, the sign of the moderated t-statistic obtained via eBayes and topTable determined gene directionality. For all models regardless of balancing, gene importance was defined by the magnitude of the t-statistic.

(4) lmgb: The present inventors used the limma package as described in #3, additionally correcting for gb-ratio (described above in the Data generation and summarization section).

(5) glmnet: The inventors applied the glmnet package (v1.9-8) (60) to CPMs, RPKMs, or voom-transformed raw counts (as in #3) to identify DE genes associated with the presence of PMLs. For balanced signatures, gene directionality was determined by the sign of the t-statistic obtained via limma by running a linear model described in #3. The inventors carried out the following series of steps using all genes for unbalanced signatures and separately using up- and down-regulated genes for balanced signatures: First, RPKMs and CPMs were z-score normalized, while raw counts were voom-transformed. Then, due to the binary character of our response variable (dysplasia status), a logistic regression model was fit using the binomial distribution family and elastic net mixing parameter α=0.5 (indicating a tradeoff between ridge and lasso regressions). The standardize option was set to FALSE, causing the coefficients to be returned on the original scale, thus allowing their magnitude to be interpreted as gene importance. Next, a range of regularization parameters λ was generated via leave-one-out cross-validation (nfolds=46), and the λ giving the minimum mean cross-validated error (lambda.min) was chosen to estimate the coefficients. Finally, DE genes were defined as having non-zero coefficients and then sorted by importance based on the coefficients' magnitude.

(6) randomForest: The inventors applied the randomForest package (v4.6-12) (61) to CPMs, RPKMs, and voom-transformed raw counts (as in #3), setting the number of trees (ntree) to 100 and importance to TRUE. For balanced signatures, the sign of the t-statistic as described in #5 determined gene directionality. For all models regardless of balancing, gene importance was determined by the magnitude of the importance variable, defined as the mean decrease in accuracy over both conditions.

(7) DESeq: The inventors applied the DESeq package (v1.18.0) (62) to unmodified raw counts only. DE analysis to find genes associated with the presence of PMLs included data normalization (estimation of the effective library size), variance estimation, and inference for two experimental conditions, as outlined in the DESeq package vignette (bioconductor.org/packages/3.3/bioc/vignettes/DESeq/inst/doc/DESeq.pdf). For balanced signatures, the sign of the log 2-fold change of expression between the two conditions determined gene directionality. For all models regardless of balancing, gene importance was defined by FDR.

(8) SVA: The inventors applied the sva package (v3.12.0) (37) to CPMs, RPKMs, or voom-transformed raw counts. Raw counts were voom-transformed using a linear model including only dysplasia status as the predictor variable. The number of surrogate variables (SVs) not associated with dysplasia status was estimated using the default approach of Buja and Eyuboglu (63) (“be” method). SVs were then identified using the empirical estimation of control probes (“irw” method), and up to 5 were added as covariates in the linear model (limma package). The adjusted model was then used to once again voom-transform raw counts, and subsequently fitted to identify DE genes associated with the presence of PMLs. For balanced signatures, the sign of the moderated t-statistic obtained via topTable determined gene directionality. For all models regardless of balancing, gene importance was defined by the magnitude of the t-statistic.

(9) pAUC (partial AUC) (64): The present inventors applied the rowpAUCs function in the genefilter package (v1.48.1) (65) to CPMs, RPKMs, or voom-transformed raw counts (as in #3). The inventors used 10 class label permutations and a sensitivity cutoff of 0.1 for a specificity range of 0.9-1. For balanced signatures, the sign of the moderated t-statistic obtained via limma's topTable determined gene directionality. For all models regardless of balancing, gene importance was defined by the magnitude of the t-statistic.

Gene signature size. After the feature selection step, the inventors selected the top scoring 10, 20, 40, 60, 80, 100, or 200 genes, making sure that for balanced signatures, half originated from an ordered list of up-regulated genes, and half from an ordered list of down-regulated genes.

Prediction method. For each set of genes, multiple prediction methods were applied to predict dysplasia status (presence of PMLs) in a training set of 46 samples and a test set of 12 samples. These training and test set samples differed in each iteration, which resulted from randomly splitting the 58 discovery set samples (FIG. 8). The following prediction methods were used:

1. glmnet: The inventors used glmnet (v1.9-8) (60) to first estimate a range of penalty parameters λ in 10-fold cross validation using the binomial distribution family parameter and α=0 to ensure all feature-selected genes were included in predictions. Dysplasia status was then predicted as a binary class, using lambda.min penalty.

2. wv (weighted voting) (55): Weighted voting algorithm was used to predict dysplasia status.

3. svm (Support Vector Machine) (66): The inventors used the svm function in the e1071 package (v1.6-7) (66) with linear kernel and 5-fold cross validation for class prediction.

4. rf (random forest): The randomForest package (v4.6-12) (61) was used with 1000 trees, requesting a matrix of class probabilities as output.

5. nb (Naïve Bayes): The naiveBayes function was used in the e1071 package (v1.6-7) with default parameters.

Each of the prediction algorithms generated a vector of predicted scores and a vector of predicted labels for all samples in the training and test sets.

Performance metrics. The present inventors considered 6,160 statistically and computationally viable combinations of the above parameters. The predicted class labels calculated for each model (i.e., a combination of parameters), coupled with true class labels were then used to calculate performance metrics for the biomarker as follows:

$\begin{matrix} \begin{matrix} {Accuracy} & \frac{{TP} + {TN}}{{TP} + {TN} + {FP} + {FN}} \\ {Sensitivity} & \frac{TP}{{TP} + {FN}} \\ {Specificity} & \frac{TN}{{FP} + {TN}} \\ {{Positive}\mspace{14mu}{Predictive}\mspace{14mu}{Value}} & \frac{TP}{{TP} + {FP}} \\ {{{Nega}{tive}}\mspace{14mu}{Predictive}\mspace{14mu}{Value}} & \frac{TN}{{TN} + {FN}} \\ {{{Matthew}'}s\mspace{14mu}{Correlation}\mspace{14mu}{Coefficient}\mspace{11mu}({MCC})} & \frac{\left( {{TP} \times TN} \right) - \left( {FP \times FN} \right)}{\sqrt{\begin{matrix} {\left( {{TP} + {FP}} \right)\left( {{TP} + {FN}} \right)} \\ {\left( {{TN} + {FP}} \right)\left( {{TN} + {FN}} \right)} \end{matrix}}} \\ {{{AUC}\mspace{14mu}{for}\mspace{14mu}{ROC}}\mspace{11mu}} & \; \\ \left( {{Receiver}\mspace{14mu}{Operating}\mspace{14mu}{Characteristic}} \right) & \; \\ {{MAQCII}\mspace{14mu}{metric}} & {{{0.5 \times {AUC}} + {{0.2}5 \times \left( {{MCC} + 1} \right)}},} \end{matrix} \\ {{{{{where}\mspace{14mu}{TP}} = {{true}\mspace{14mu}{positives}}};{{FP} = {{false}\mspace{14mu}{positives}}};}\mspace{14mu}} \\ {{{TN} = {{true}\mspace{14mu}{negativies}}};{{FN} = {{false}\mspace{14mu}{negatives}}};} \\ {{{MCC} = {{{Matthew}'}s\mspace{14mu}{Correlation}\mspace{14mu}{Coefficient}}};{and}} \\ {{AUC} = {{Area}\mspace{14mu}{Under}\mspace{14mu}{the}\mspace{14mu}{{Curve}.}}} \end{matrix}$

For each model, we calculated these metrics for each of the 500 iterations (different training and test sets assembled from the discovery set samples) and then averaged over all iterations. In addition to the standard performance metrics, we calculated model overfitting and gene selection consistency. The overfitting metric was calculated as the difference between the train set AUC and the test set AUC. Specifically, a model performing well on the training set but poorly on the test set would achieve a high overfitting score. For each model, the gene selection consistency metric was calculated as the average (“normalized” to biomarker size in a given model) percentage of genes passing the gene filter, that were selected into the final gene committee in all 500 iterations:

${consistency} = {1 - \frac{\begin{matrix} {{\#\mspace{14mu}{unique}\mspace{14mu}{genes}\mspace{14mu}{in}\mspace{14mu}{all}\mspace{14mu}{iterations}} -} \\ {{biomarker}\mspace{14mu}{size}} \end{matrix}}{\begin{matrix} {\left( {{biomarker}\mspace{14mu}{size} \times \#\mspace{14mu}{iterations}} \right) -} \\ {{biomarker}\mspace{14mu}{size}} \end{matrix}}}$

For example, a model requiring a 10-gene biomarker would have the highest consistency (1) if it selected the same 10 genes in all 500 iterations (10 unique genes selected altogether). The same model would have the lowest consistency (0) if it selected a different set of 10 genes in all iterations (10 genes×500 iterations=5000 unique genes altogether).

Selection of best model. In selecting the best model from among the 6,160 the inventors tested and considered the degree of model overfitting, model gene selection consistency and test set AUC. First, top 10% (n=616) least overfitting models were identified. Simultaneously, the inventors identified top 10% (n=616) most consistent models. Finally, the model with the highest test set AUC among models fulfilling both criteria (n=121) was chosen as the final model.

Selection of final gene signature. The biomarker genes selected may differ between iterations due to changes in the training set. Therefore, to generate a final gene signature, the inventors trained the biomarker using all 58 discovery set samples and best model parameters.

Positive and negative controls. The biomarker discovery pipeline was also used to develop control biomarkers. As positive controls, the inventors used smoking status and sex phenotypes to identify biomarkers that could successfully distinguish former from current smokers (AUC=0.99), and females from males (AUC=0.96). As negative controls, the inventors used randomly shuffled labels for dysplasia status (AUC=0.48), smoking status (AUC=0.52), and sex (AUC=0.51). Label shuffling was conducted preserving the association between gene expression profiles and remaining phenotypes; i.e., in the case of shuffled dysplasia status, only dysplasia status was shuffled while other phenotypes and the corresponding gene expression profile remained unchanged and linked to the same sample ID.

Validations. The performance of the final biomarker was tested using the biomarker discovery pipeline in validation mode. In this mode, the pipeline takes in the entire discovery set (n=58) as the training set, and an external validation set as the test set. The test set is first corrected for gb-ratio (RNA-Seq quality metric) using limma, and the residual data is used as input. Both training and test sets are then z-score normalized. The pipeline was run using only the final model to generate prediction labels and prediction scores for the test set samples. Finally, pROC package (v1.8) (67) was used to visualize and quantify biomarker performance by plotting a ROC curve using prediction scores as the response and the dichotomous phenotype as the predictor, and extracting the AUC value from the resulting ROC object.

Detecting PML Presence in Validation Set Samples

In order to validate the biomarker's ability to detect the presence of PMLs, the performance of the biomarker was tested in smokers with and without PMLs (n=17 samples) left out of the biomarker discovery process. To assess the robustness of the results, we randomly permuted dysplasia status labels 100 times, obtaining biomarker scores for all 17 samples in each of the iterations. The present inventors then concatenated the 100 newly generated biomarker score sets for randomized labels, creating a predictor vector consisting of 1700 scores. Similarly, the inventors concatenated 100 identical copies of biomarker score sets for true labels, creating a response vector of the same length. This allowed the inventors to visualize the performance of the biomarker on true and randomized labels in a single ROC curve (FIG. 5).

Predicting PML Progression in Longitudinally-Collected Samples

In order to validate the biomarker's ability to predict sample progression/regression, the present inventors first used the biomarker to score the longitudinally collected RPCI samples (n=51). Next, calculated the difference in scores between two consecutive time points were calculated for each patient (later time point biomarker score−earlier time point biomarker score). For example, a subject with 3 samples from 3 different time points would have 3 scores, and thus two score differences could be calculated; a subject with 2 samples from 2 time points would have 2 scores, and thus 1 score difference.

Each pair of samples was assigned a “progressing/stable” or “regressing” phenotype. A “progressing/stable” phenotype indicated that the worst histological grade of PMLs sampled during the baseline procedure increased in severity or remained unchanged at follow-up; while a “regressing” phenotype indicated that the worst histological grade of PMLs sampled at baseline decreased in severity at follow-up.

The ability of the score difference to predict the “progression/regression” phenotype was quantified by plotting a ROC curve, using the vector of score differences as the predictor variable, and the progression/regression phenotype as the response variable.

Implementation of the method. The framework and structure of this pipeline are based on principles outlined for microarray data applications. The pipeline outlined in this paper was substantially modified to accommodate RNA-Seq data as well as RNA-Seq-specific methods.

Subject Inclusion/Exclusion Criteria for Samples from the British Columbia Cancer Agency (BCCA)

The samples with normal/hyperplasia histology are part of the Pan-Canadian Study and included subjects between 50 and 75 years old, current or former smokers who have smoked cigarettes for 20 years or more, and that had an estimated 3-year lung cancer risk of greater than or equal to 2%. Exclusion criteria included medical conditions, such as severe heart disease, that would jeopardize the subject's safety during participation in the study, previously diagnosed lung cancer, ex-smokers of greater than or equal to 15 years, anti-coagulant treatment, and pregnancy. The subjects with airway dysplasia were participants in three different chemoprevention studies for green tea extract (n=27 samples), sulindac (n=4 samples), and myo-inositol (n=13 samples) or from the Pan-Canadian Study described above (n=6). All samples were collected at the BCCA at baseline prior to administration of therapeutic interventions. Inclusion criteria for these chemoprevention trials can be summarized as subjects between 40 and 79 years of age, current or former smokers with at least 30 pack-years, no lung cancer history or stage 0/I curatively treated NSCLC either at least 1 year or 6 months prior to the trial (depending on trial). Exclusion criteria varied by trial but included medical conditions that would jeopardize the subject's safety during participation of the study and pregnancy. See details below:

Green Tea: Inclusion Criteria

-   -   Women or men age 45 to 74 years of age     -   Current or former smokers who have smoked at least 30         pack-years, e.g. 1 pack per day for 30 years or more (a former         smoker is defined as one who has stopped smoking for one or more         years)     -   ECOG performance status 0 or 1     -   C-Reactive Protein >1.2 mg/L     -   One or more areas of dysplasia with a surface diameter larger         than 1.2 mm on autofluorescence bronchoscopy     -   Willing to take Polyphenon E/placebo twice a day regularly     -   Since it is unknown if Polyphenon E or EGCG will cause fetal         harm when administered during pregnancy, women subjects must be         postmenopausal (no menstrual periods >1 year or elevated FSH >40         mIU/ml), surgically sterile, or using birth control pill. Women         of childbearing age must have normal β-HCG within 14 days to         exclude pregnancy.     -   Normal renal and liver function defined as serum creatinine         bilirubin, AST, ALT or alkaline phosphatase levels below the         upper limit of normal     -   Agreeing to sign, on initial interview, informed consent forms         for screening procedures (sputum cytometry analysis,         fluorescence bronchoscopy, and low dose spiral thoracic CT         scan). Once eligibility has been determined for the         chemoprevention trial participation, agreeing to sign a         study-specific treatment informed consent form.

Exclusion Criteria

-   -   Consumption of more than 7 cups of tea a week     -   Use of other natural health products containing green tea         compounds     -   Chronic active hepatitis/liver cirrhosis     -   Severe heart disease, e.g. unstable angina, chronic congestive         heart failure, use of antiarrhythmic agents     -   Ongoing gastric ulcer     -   Have on-going rectal bleeding     -   Have a history of chronic diverticulitis and/or colitis     -   Experiencing symptoms of gastritis or hemorrhoids in which         medical treatment is required     -   Experiencing any symptomatic gastrointestinal condition that may         predispose the individual to gastrointestinal bleeding     -   Acute bronchitis or pneumonia within one month     -   Carcinoma in-situ or invasive cancer on bronchoscopy or abnormal         spiral chest CT suspicious of lung cancer     -   Known reaction to Xylocaine salbutamol, midazolam, and         alfentanil     -   Known allergy to green tea and/or corn starch, gelatin, or other         nonmedicinal ingredients     -   Any medical condition, such as acute or chronic respiratory         failure, or bleeding disorder, that in the opinion of the         investigator could jeopardize the subject's safety during         participation in the study     -   On anti-coagulant treatment such as warfarin or heparin     -   Breastfeeding     -   Pregnancy     -   Unwilling to have a bronchoscopy     -   Unwilling to have a spiral chest CT     -   Unwilling to sign a consent

Sulindac: Inclusion Criteria

-   -   Men and women 40 through 79 years of age     -   Current or former smokers with a >30 pack-year smoking history         and (a) no prior lung cancer, (b) stage I NSCLC resected at         least one year prior to Registration/Randomization, or (c) stage         I Non-Small Cell Lung Cancer (NSCLC) with a >1 year interval         since adjuvant chemotherapy conclusion     -   Women of childbearing potential and men must agree to use         adequate contraception (hormonal or barrier method of birth         control; abstinence) prior to study entry and for the duration         of study participation. Should a woman become pregnant or         suspect she is pregnant while participating in this study, she         should inform her treating physician immediately.     -   A negative (serum or urine) pregnancy test done <7 days prior to     -   Registration/Randomization, for women of childbearing potential         only     -   Willingness to provide tissue blocks and sputum samples for         research purposes     -   Participants must have normal organ and marrow function as         defined below and obtained ≤45 days prior to         Registration/Randomization:     -   Hemoglobin ≥lower limit of institutional normal (LLN)     -   Leukocytes ≥3,000/μL     -   Absolute neutrophil count ≥1,500/μL     -   Platelets ≥100,000/μL     -   Direct bilirubin ≤1.5×institutional upper limit of normal (ULN)     -   ALT (SGPT)≤1.5×institutional ULN     -   Creatinine ≤1.5×institutional ULN or calculated creatinine         clearance ≥30 ml/min     -   ≥1 site of histologically-confirmed bronchial dysplasia     -   ECOG performance status ≤1     -   Negative chest x-ray     -   Negative electrocardiogram

Exclusion Criteria

-   -   Prior history of cancer (within the previous 3-years).         Exception: Stage I NSCLC as outlined above, nonmelanomatous skin         cancer, localized prostate cancer, carcinoma in situ (CIS) of         cervix, or superficial bladder cancer with conclusion of         treatment >6 months prior to Registration/Randomization.     -   Prior pneumonectomy     -   Solid organ transplant recipients     -   History of GI ulceration, bleeding or perforation     -   Uncontrolled intercurrent illness including, but not limited to:         ongoing or active infection, symptomatic congestive heart         failure, unstable angina pectoris, cardiac arrhythmia, recent         (<6 months) history of MI, chronic renal disease, chronic liver         disease, difficult to control hypertension or psychiatric         illness/social situations that would limit compliance with study         requirements.     -   Recent (<6 months) participation in another chemoprevention         trial     -   Participant currently receiving any other investigational agents     -   Any supplemental oxygen use (continuous or intermittent use) or         documented     -   Room Air (RA) SaO2<90%     -   Pregnant women. Note: because there are no adequate,         well-controlled studies in pregnant women and sulindac is         absolutely contraindicated in the 3rd trimester.     -   Breastfeeding women. Note: because there is an unknown but         potential risk for adverse events in nursing infants secondary         to treatment of the mother with sulindac, women who are         breast-feeding will be excluded.     -   Individuals who are known to be HIV positive. Note: HIV positive         individuals are excluded for the following two reasons. First,         HIV positive individuals are known to have altered immune         function. Since one of the potential mechanisms of action of         sulindac is proposed to be enhancement of immune function in         preventing lung cancer progression, it is not known how the         presence of HIV infection would alter this enhancement of immune         function as compared to non-HIV infected individuals. Second,         individuals with HIV are also known to be at higher risk for         lung cancer then non-HIV infected individuals which would alter         the risk/incidence of lung cancer in our study population.     -   Regular NSAID or corticosteroid use during the 6-month period         prior to intervention (may be eligible after washout period of         12 weeks for NSAIDs and 6 weeks for corticosteroids)     -   Regular aspirin use. Exception: Aspirin can be used if         prescribed by a physician for prevention. Maximum of one aspirin         (81 mg) per day allowed.     -   History of allergic reactions or hypersensitivity to sulindac or         other NSAIDS, including aspirin-sensitive asthma     -   Women of childbearing potential who are unwilling to employ         adequate contraception (hormonal or barrier method of birth         control; abstinence) prior to study entry and for the duration         of study participation. Note: Effects of sulindac on the         developing human fetus at the recommended therapeutic dose are         fetal harm early in pregnancy. However, there are known harmful         adverse events in the third trimester of pregnancy. Should a         woman become pregnant or suspect she is pregnant while         participating in this study, she should inform her treating         physician immediately.     -   Current use of methotrexate, corticosteroids, (anti-platelet         agents) warfarin, ticlopidine, clopidogrel, aspirin, abciximab,         dipyridamole, eptifibatide, tirofiban, lithium, cyclosporine,         hydralazine, ACE inhibitors

Myo-Inositol: Inclusion Criteria

-   -   Ability to understand and willingness to sign a written informed         consent document     -   Age ≥45 to ≤79     -   ECOG performance status (PS) 0 or 1     -   One or both of the following: Stage 0/I curatively treated         non-small cell lung cancer (NSCLC) with a ≥30 pack-year smoking         history (surgery, adjuvant chemotherapy or radiotherapy must be         completed ≥6 months prior to screening); OR Current or former         smokers with a ≥30 pack-year smoking history without a history         of lung cancer. Pack-years is determined by multiplying the         number of packs smoked per day by the number of years smoked.     -   Women of childbearing capacity who agree to use an acceptable         form of birth control for the duration of the study (e.g.         condom, oral contraceptives, etc.)

Exclusion Criteria

-   -   Prior history of cancer, with the following exceptions:     -   ≥3-year disease free interval (with the exception of stage I         NSCLC as described above)     -   Non-melanomatous skin cancer     -   Localized prostate cancer with conclusion of treatment ≥6 months         prior to screening     -   Carcinoma in situ (CIS) of cervix with conclusion of treatment         ≥6 months prior to screening     -   Superficial bladder cancer with conclusion of treatment ≥6         months prior to screening     -   Prior pneumonectomy     -   Solid organ transplant recipients     -   Uncontrolled intercurrent illness including, but not limited to:         ongoing or active infection, symptomatic congestive heart         failure, unstable angina pectoris, cardiac arrhythmia, severe         chronic obstructive pulmonary disease requiring supplemental         oxygen, difficult to control hypertension, or psychiatric         illness/social situations that would limit compliance with study         requirements.     -   Schizophrenia     -   Bipolar disorder     -   Lithium treatment     -   Carbamazepine treatment     -   Valproate treatment     -   Diabetes     -   Currently using other natural health products containing         inositol     -   Anticoagulant use such as Coumadin or heparin. Exception:         participant is off those drugs for ≥7 days prior to         pre-registration.     -   Recent (≤6 months) participation in another chemoprevention         trial     -   Participant currently receiving any other investigational agents     -   Any supplemental oxygen use (continuous or intermittent use) or         documented Room Air (RA) SaO₂<90%     -   Pregnant women. (Excluded because the effects of high doses of         myo-inositol on the fetus or newborn are not known.)     -   Breastfeeding women. (Excluded because the risk for adverse         events in nursing infants secondary to treatment of the mother         with high doses of myo-inositol are not known.)     -   History of allergic reactions attributed to myo-inositol     -   History of allergies to any ingredient in the study product or         placebo

Early Detection of Lung Cancer—A Pan-Canadian Study: Inclusion Criteria

-   -   Women or men age 50 to 75 years     -   Current or former smokers who have smoked cigarettes for 20         years or more (a former smoker is defined as one who has stopped         smoking for one or more years)     -   An estimated 3-year lung cancer risk of >2% based on the risk         prediction model.     -   ECOG performance status 0 or 1     -   Capable of providing, informed consent for screening procedures         (low dose spiral CT, AFB, spirometry, blood biomarkers)

Exclusion Criteria

-   -   Any medical condition, such as severe heart disease (e.g.         unstable angina, chronic congestive heart failure), acute or         chronic respiratory failure, bleeding disorder, that in the         opinion of the investigator could jeopardize the subject's         safety during participation in the study or unlikely to benefit         from screening due to shortened life-expectancy from the         co-morbidities     -   Have been previously diagnosed with lung cancer     -   Have had other cancer with the exception of the following         cancers which can be included in the study: non-melanomatous         skin cancer, localized prostate cancer, carcinoma in situ (CIS)         of the cervix, or superficial bladder cancer. Treatment of the         exceptions must have ended >6 months before registration into         this study.     -   Ex-smoker for ≥15 years     -   On anti-coagulant treatment such as warfarin or heparin     -   Known reaction to Xylocaine, salbutamol, midazolam, and         alfentanil     -   Pregnancy     -   Unwilling to have a spiral chest CT     -   Chest CT within 2 years     -   Unwilling to sign a consent         Subject Inclusion/Exclusion Criteria for Samples from RPCI

Subjects met the following high-risk lung screening criteria: 1) Personal cancer history of the lung, bronchus, head/neck, and/or esophagus and no evidence of disease at the time of enrollment, or 2.) No personal history of upper aerodigestive cancer, age 50+, and a current smoker or a former smoker with 20+ pack years. In addition, subjects in the second group had to have one or more risk factors including chronic lung disease such as emphysema, chronic bronchitis, or chronic obstructive pulmonary disease, occupationally related asbestos disease, or a family history of lung cancer in a first degree relative.

TABLE 1 Demographic and clinical characteristics stratified by premalignant lesion status. Data are means (SD) for continuous variables and proportions with percentages for Overall No Lesions Lesions Factor (n = 82) (n = 25) (n = 50) P* Age 62.9 (7.2) 64.5 (5.8) 62.2 (8.0) 0.16 Male 54/82 (65.9) 16/25 (64) 35/50 (70) 0.61 Current smoker 40/82 (48.8) 11/25 (44) 25/50 (50) 0.81 Pack-years 47.3 (15.7) 47.6 (17.9) 47.2 (15.2) 0.93 FEV1% Predicted 82.5 (18.6) 84.5 (17.9) 81.7 (19.2) 0.54 FEV1/FVC Ratio 71.2 (7.9) 73.4 (7.4) 69.6 (8.1) 0.05 COPD (FEV1% < 80 & 24/82 (29.3) 5/25 (20) 17/50 (34) 0.28 FEV1/FVC < 70) Histology <10.001 Normal 12/82 (14.6) 12/25 (48) Hyperplasia 13/82 (15.9) 13/25 (52) Metaplasia 7/82 (8.5) Mild Dysplasia 35/82 (42.7) 35/50 (70) Moderate Dysplasia 12/82 (14.6) 12/50 (24) Severe Dysplasia 3/82 (3.7) 3/50 (6) dichotomous variables. P* values are for the comparison of subjects with and without premalignant lesions. Two sample t-tests were used for continuous variables; Fisher's exact test was used for categorical variables.

TABLE 2 Alignment statistics stratified by premalignant lesion status Data are means (SD) for continuous variables and proportions with percentages for dichotomous variables. Reads are expressed in millions denoted by M. P* values are Overall No Lesions Lesions Factor (n = 82) (n = 25) (n = 50) P* Total Alignments 90M (17M) 90M (15M) 91M (19M) 0.78 Unique Alignments 83M (16M) 83M (14M) 84M (17M) 0.76 Properly Paired 66M (12M) 66M (11M) 67M (14M) 0.75 Alignments Genebody 80/20 Ratio 1.3 (0.2) 1.3 (0.1) 1.3 (0.2) 0.84 Mean GC Content 47.8 (3.4) 47.4 (2.9) 48.2 (3.7) 0.34 for the comparison of subjects with and without premalignant lesions. Two sample t-tests were used for continuous variables; Fisher's exact test was used for factors.

TABLE 3 280 genes differentially expressed between subjects with PMLs and without PMLs Ensembl entrezgene hgnc_symbol gene_biotype wikigene_description Direction ENSG00000223959 172 AFG3L1P pseudogene AFG3 ATPase Down-regulated in family gene 3-like the presence of 1 (S. cerevisiae), dyplasia pseudogene ENSG00000115282 64427 TTC31 protein_coding tetratricopeptide Down-regulated in repeat domain 31 the presence of dyplasia ENSG00000139631 51380 CSAD protein_coding cysteine sulfinic Down-regulated in acid decarboxylase the presence of dyplasia ENSG00000198198 23334 SZT2 protein_coding seizure threshold 2 Down-regulated in homolog (mouse) the presence of dyplasia ENSG00000167524 124923 protein_coding uncharacterized Down-regulated in serine/threonine- the presence of protein kinase dyplasia SgK494 ENSG00000242028 25764 Cl5orf63 protein_coding chromosome 15 Down-regulated in open reading frame the presence of 63 dyplasia ENSG00000235194 NA PPP1R3E protein_coding Down-regulated in the presence of dyplasia ENSG00000179979 285464 CRIPAK protein_coding cysteine-rich PAK1 Down-regulated in inhibitor the presence of dyplasia ENSG00000164970 203259 FAM219A protein_coding family with Down-regulated in sequence similarity the presence of 219, member A dyplasia ENSG00000162231 10482 NXF1 protein_coding nuclear RNA Down-regulated in export factor 1 the presence of dyplasia ENSG00000010322 11188 NISCH protein_coding nischarin Down-regulated in the presence of dyplasia ENSG00000121310 55268 ECHDC2 protein_coding enoyl CoA Down-regulated in hydratase domain the presence of containing 2 dyplasia ENSG00000167978 23524 SRRM2 protein_coding serine/arginine Down-regulated in repetitive matrix 2 the presence of dyplasia ENSG00000229180 NA lincRNA Down-regulated in the presence of dyplasia ENSG00000108799 2145 EZH1 protein_coding enhancer of zeste Down-regulated in homolog 1 the presence of (Drosophila) dyplasia ENSG00000070476 79364 ZXDC protein_coding ZXD family zinc Down-regulated in finger C the presence of dyplasia ENSG00000186088 54103 PION protein_coding pigeon homolog Down-regulated in (Drosophila) the presence of dyplasia ENSG00000132680 22889 KIAA0907 protein_coding KIAA0907 Down-regulated in the presence of dyplasia ENSG00000122965 9904 RBM19 protein_coding RNA binding motif Down-regulated in protein 19 the presence of dyplasia ENSG00000130766 83667 SESN2 protein_coding sestrin 2 Down-regulated in the presence of dyplasia ENSG00000064607 10147 SUGP2 protein_coding SURP and G patch Down-regulated in domain containing the presence of 2 dyplasia ENSG00000184863 155435 RBM33 protein_coding RNA binding motif Down-regulated in protein 33 the presence of dyplasia ENSG00000214021 26140 TTLL3 protein_coding tubulin tyrosine Down-regulated in ligase-like family, the presence of member 3 dyplasia ENSG00000080603 10847 SRCAP protein_coding Snf2-related Down-regulated in CREBBP activator the presence of protein dyplasia ENSG00000072121 23503 ZFYVE26 protein_coding zinc finger, FYVE Down-regulated in domain containing the presence of 26 dyplasia ENSG00000182873 NA antisense Down-regulated in the presence of dyplasia ENSG00000104365 3551 IKBKB protein_coding inhibitor of kappa Down-regulated in light polypeptide the presence of gene enhancer in dyplasia B-cells, kinase beta ENSG00000167522 29123 ANKRD11 protein_coding ankyrin repeat Down-regulated in domain 11 the presence of dyplasia ENSG00000213190 10962 MLLT11 protein_coding myeloid/lymphoid Down-regulated in or mixed-lineage the presence of leukemia (trithorax dyplasia homolog, Drosophila); translocated to, 11 ENSG00000135407 10677 AVIL protein_coding advillin Down-regulated in the presence of dyplasia ENSG00000185219 353274 ZNF445 protein_coding zinc finger protein Down-regulated in 445 the presence of dyplasia ENSG00000163486 23380 SRGAP2 protein_coding SLIT-ROBO Rho Down-regulated in GTPase activating the presence of protein 2 dyplasia ENSG00000087266 6452 SH3BP2 protein_coding SH3-domain Down-regulated in binding protein 2 the presence of dyplasia ENSG00000198563 692199 DDX39B protein_coding DEAD (Asp-Glu- Down-regulated in Ala-Asp) box the presence of polypeptide 39B dyplasia ENSG00000142528 25888 ZNF473 protein_coding zinc finger protein Down-regulated in 473 the presence of dyplasia ENSG00000123064 79039 DDX54 protein_coding DEAD (Asp-Glu- Down-regulated in Ala-Asp) box the presence of polypeptide 54 dyplasia ENSG00000042062 140876 FAM65C protein_coding family with Down-regulated in sequence similarity the presence of 65, member C dyplasia ENSG00000247484 NA NA NA NA Down-regulated in the presence of dyplasia ENSG00000100201 10521 DDX17 protein_coding DEAD (Asp-Glu- Down-regulated in Ala-Asp) box the presence of helicase 17 dyplasia ENSG00000125633 54520 CCDC93 protein_coding coiled-coil domain Down-regulated in containing 93 the presence of dyplasia ENSG00000257479 NA lincRNA Down-regulated in the presence of dyplasia ENSG00000076108 11176 BAZ2A protein_coding bromodomain Down-regulated in adjacent to zinc the presence of finger domain, 2A dyplasia ENSG00000137221 93643 TJAP1 protein_coding tight junction Down-regulated in associated protein 1 the presence of (peripheral) dyplasia ENSG00000215424 114044 MCM3 lincRNA MCM3AP Down-regulated in AP-AS1 antisense RNA 1 the presence of (non-protein dyplasia coding) ENSG00000100941 5411 PNN protein_coding pinin, desmosome Down-regulated in associated protein the presence of dyplasia ENSG00000170949 90338 ZNF160 protein_coding zinc finger protein Down-regulated in 160 the presence of dyplasia ENSG00000240053 58496 LY6G5B protein_coding lymphocyte antigen Down-regulated in 6 complex, locus the presence of G5B dyplasia ENSG00000181523 6448 SGSH protein_coding N- Down-regulated in sulfoglucosamine the presence of sulfohydrolase dyplasia ENSG00000131398 3748 KCNC3 protein_coding potassium voltage- Down-regulated in gated channel, the presence of Shaw-related dyplasia subfamily, member 3 ENSG00000129933 23383 MAU2 protein_coding MAU2 chromatid Down-regulated in cohesion factor the presence of homolog (C. dyplasia elegans) ENSG00000161010 51149 C5orf45 protein_coding chromosome 5 Down-regulated in open reading frame the presence of 45 dyplasia ENSG00000110888 65981 CAPRIN2 protein_coding caprin family Down-regulated in member 2 the presence of dyplasia ENSG00000130254 9667 SAFB2 protein_coding scaffold attachment Down-regulated in factor B2 the presence of dyplasia ENSG00000184634 9968 MED12 protein_coding mediator complex Down-regulated in subunit 12 the presence of dyplasia ENSG00000077157 4660 PPP1R12B protein_coding protein phosphatase Down-regulated in 1, regulatory the presence of subunit 12B dyplasia ENSG00000133624 79970 ZNF767 pseudogene zinc finger family Down-regulated in member 767 the presence of dyplasia ENSG00000227372 57212 TP73-AS1 lincRNA TP73 antisense Down-regulated in RNA 1 (non- the presence of protein coding) dyplasia ENSG00000100813 22985 ACIN1 protein_coding apoptotic Down-regulated in chromatin the presence of condensation dyplasia inducer 1 ENSG00000127511 23309 SIN3B protein_coding SIN3 transcription Down-regulated in regulator homolog the presence of B (yeast) dyplasia ENSG00000155363 4343 MOV10 protein_coding Mov10, Moloney Down-regulated in leukemia virus 10, the presence of homolog (mouse) dyplasia ENSG00000124222 8675 STX16 protein_coding syntaxin 16 Down-regulated in the presence of dyplasia ENSG00000099331 4650 MYO9B protein_coding myosin IXB Down-regulated in the presence of dyplasia ENSG00000169246 NA NPIPL3 protein_coding Down-regulated in the presence of dyplasia ENSG00000137343 79969 ATAT1 protein_coding alpha tubulin Down-regulated in acetyltransferase 1 the presence of dyplasia ENSG00000169045 3187 HNRNPH1 protein_coding heterogeneous Down-regulated in nuclear the presence of ribonucleoprotein dyplasia H1 (H) ENSG00000205047 NA protein_coding Down-regulated in the presence of dyplasia ENSG00000198853 9853 RUSC2 protein_coding RUN and SH3 Down-regulated in domain containing the presence of 2 dyplasia ENSG00000197375 6584 SLC22A5 protein_coding solute carrier Down-regulated in family 22 (organic the presence of cation/carnitine dyplasia transporter), member 5 ENSG00000182796 440104 TMEM198B pseudogene transmembrane Down-regulated in protein 198B, the presence of pseudogene dyplasia ENSG00000182944 2130 EWSR1 protein_coding Ewing sarcoma Down-regulated in breakpoint region 1 the presence of dyplasia ENSG00000065526 23013 SPEN protein_coding spen homolog, Down-regulated in transcriptional the presence of regulator dyplasia (Drosophila) ENSG00000137337 9656 MDC1 protein_coding mediator of DNA- Down-regulated in damage checkpoint the presence of 1 dyplasia ENSG00000186174 283149 BCL9L protein_coding B-cell Down-regulated in CLL/lymphoma 9- the presence of like dyplasia ENSG00000075568 23505 TMEM131 protein_coding transmembrane Down-regulated in protein 131 the presence of dyplasia ENSG00000170322 4798 NFRKB protein_coding nuclear factor Down-regulated in related to kappaB the presence of binding protein dyplasia ENSG00000171456 171023 ASXL1 protein_coding additional sex Down-regulated in combs like 1 the presence of (Drosophila) dyplasia ENSG00000044446 5256 PHKA2 protein_coding phosphorylase Down-regulated in kinase, alpha 2 the presence of (liver) dyplasia ENSG00000166436 9866 TRIM66 protein_coding tripartite motif Down-regulated in containing 66 the presence of dyplasia ENSG00000255847 NA antisense Down-regulated in the presence of dyplasia ENSG00000245149 100507018 lincRNA uncharacterized Down-regulated in LOC100507018 the presence of dyplasia ENSG00000253200 NA protein_coding Down-regulated in the presence of dyplasia ENSG00000100226 9567 GTPBP1 protein_coding GTP binding Down-regulated in protein 1 the presence of dyplasia ENSG00000146828 56996 SLC12A9 protein_coding solute carrier Down-regulated in family 12 the presence of (potassium/chloride dyplasia transporters), member 9 ENSG00000215769 NA protein_coding Down-regulated in the presence of dyplasia ENSG00000168297 54899 PXK protein_coding PX domain Down-regulated in containing the presence of serine/threonine dyplasia kinase ENSG00000225828 100128071 protein_coding uncharacterized Down-regulated in LOC100128071 the presence of dyplasia ENSG00000115459 84173 ELMOD3 protein_coding ELMO/CED-12 Down-regulated in domain containing the presence of 3 dyplasia ENSG00000224660 100505696 lincRNA uncharacterized Down-regulated in LOC100505696 the presence of dyplasia ENSG00000090905 27327 TNRC6A protein_coding trinucleotide repeat Down-regulated in containing 6A the presence of dyplasia ENSG00000205885 283314 antisense uncharacterized Down-regulated in LOC283314 the presence of dyplasia ENSG00000117616 57035 Clorf63 protein_coding chromosome 1 Down-regulated in open reading frame the presence of 63 dyplasia ENSG00000114841 25981 DNAH1 protein_coding dynein, axonemal, Down-regulated in heavy chain 1 the presence of dyplasia ENSG00000132382 10514 MYBBP1A protein_coding MYB binding Down-regulated in protein (P160) 1a the presence of dyplasia ENSG00000061936 6433 SFSWAP protein_coding splicing factor, Down-regulated in suppressor of the presence of white-apricot dyplasia homolog (Drosophila) ENSG00000168763 26505 CNNM3 protein_coding cyclin M3 Down-regulated in the presence of dyplasia ENSG00000214765 641977 SEPT7P2 pseudogene septin 7 Down-regulated in pseudogene 2 the presence of dyplasia ENSG00000119321 23307 FKBP15 protein_coding FK506 binding Down-regulated in protein 15, 133 kDa the presence of dyplasia ENSG00000047056 22884 WDR37 protein_coding WD repeat domain Down-regulated in 37 the presence of dyplasia ENSG00000165699 7248 TSC1 protein_coding tuberous sclerosis 1 Down-regulated in the presence of dyplasia ENSG00000168970 100137047 JMJD7- protein_coding JMJD7-PLA2G4B Down-regulated in PLA2G4B readthrough the presence of dyplasia ENSG00000079277 8569 MKNK1 protein_coding MAP kinase Down-regulated in interacting the presence of serine/threonine dyplasia kinase 1 ENSG00000115568 7701 ZNF142 protein_coding zinc finger protein Down-regulated in 142 the presence of dyplasia ENSG00000167615 114823 LENG8 protein_coding leukocyte receptor Down-regulated in cluster (LRC) the presence of member 8 dyplasia ENSG00000100083 26088 GGA1 protein_coding golgi-associated, Down-regulated in gamma adaptin ear the presence of containing, ARF dyplasia binding protein 1 ENSG00000139436 9815 GIT2 protein_coding G protein-coupled Down-regulated in receptor kinase the presence of interacting ArfGAP dyplasia 2 ENSG00000168066 7536 SF1 protein_coding splicing factor 1 Down-regulated in the presence of dyplasia ENSG00000099917 51586 MED15 protein_coding mediator complex Down-regulated in subunit 15 the presence of dyplasia ENSG00000091831 2099 ESR1 protein_coding estrogen receptor 1 Down-regulated in the presence of dyplasia ENSG00000234420 100129482 ZNF37BP pseudogene zinc finger protein Down-regulated in 37B, pseudogene the presence of dyplasia ENSG00000178971 80169 CTC1 protein_coding CTS telomere Down-regulated in maintenance the presence of complex dyplasia component 1 ENSG00000114982 55683 KANSL3 protein_coding KAT8 regulatory Down-regulated in NSL complex the presence of subunit 3 dyplasia ENSG00000148840 23082 PPRC1 protein_coding peroxisome Down-regulated in proliferator- the presence of activated receptor dyplasia gamma, coactivator-related 1 ENSG00000112941 11044 PAPD7 protein_coding PAP associated Down-regulated in domain containing the presence of 7 dyplasia ENSG00000143624 65123 INTS3 protein_coding integrator complex Down-regulated in subunit 3 the presence of dyplasia ENSG00000139990 8816 DCAF5 protein_coding DDB1 and CUL4 Down-regulated in associated factor 5 the presence of dyplasia ENSG00000100650 6430 SRSF5 protein_coding serine/arginine-rich Down-regulated in splicing factor 5 the presence of dyplasia ENSG00000133460 66035 SLC2A11 protein_coding solute carrier Down-regulated in family 2 (facilitated the presence of glucose dyplasia transporter), member 11 ENSG00000102125 6901 TAZ protein_coding tafazzin Down-regulated in the presence of dyplasia ENSG00000136828 9649 RALGPS1 protein_coding Ral GEF with PH Down-regulated in domain and SH3 the presence of binding motif 1 dyplasia ENSG00000235027 NA antisense Down-regulated in the presence of dyplasia ENSG00000235706 400242 DICER1- lincRNA DICER1 antisense Down-regulated in AS1 RNA 1 (non- the presence of protein coding) dyplasia ENSG00000205890 100128770 antisense uncharacterized Down-regulated in LOC100128770 the presence of dyplasia ENSG00000133943 80017 C14orf159 protein_coding chromosome 14 Down-regulated in open reading frame the presence of 159 dyplasia ENSG00000100068 91355 LRP5L protein_coding low density Down-regulated in lipoprotein the presence of receptor-related dyplasia protein 5-like ENSG00000234616 NA JRK processed_ Down-regulated in transcript the presence of dyplasia ENSG00000115687 23178 PASK protein_coding PAS domain Down-regulated in containing the presence of serine/threonine dyplasia kinase ENSG00000243335 154881 KCTD7 protein_coding RAB guanine Down-regulated in nucleotide the presence of exchange factor dyplasia (GEF) 1 ENSG00000131149 23199 KIAA0182 protein_coding KIAA0182 Down-regulated in the presence of dyplasia ENSG00000184677 9923 ZBTB40 protein_coding zinc finger and Down-regulated in BTB domain the presence of containing 40 dyplasia ENSG00000116580 54856 GON4L protein_coding gon-4-like (C. Down-regulated in elegans) the presence of dyplasia ENSG00000130684 26152 ZNF337 protein_coding zinc finger protein Down-regulated in 337 the presence of dyplasia ENSG00000143442 23126 POGZ protein_coding pogo transposable Down-regulated in element with ZNF the presence of domain dyplasia ENSG00000249093 NA NA NA NA Down-regulated in the presence of dyplasia ENSG00000173064 283450 C12orf51 protein_coding chromosome 12 Down-regulated in open reading frame the presence of 51 dyplasia ENSG00000215039 678655 lincRNA uncharacterized Down-regulated in LOC678655 the presence of dyplasia ENSG00000178038 259173 ALS2CL protein_coding ALS2 C-terminal Down-regulated in like the presence of dyplasia ENSG00000258461 NA processed_ Down-regulated in transcript the presence of dyplasia ENSG00000146830 64599 GIGYF1 protein_coding GRB10 interacting Down-regulated in GYF protein 1 the presence of dyplasia ENSG00000234290 NA antisense Down-regulated in the presence of dyplasia ENSG00000120318 64411 ARAP3 protein_coding ArfGAP with Down-regulated in RhoGAP domain, the presence of ankyrin repeat and dyplasia PH domain 3 ENSG00000162241 283130 SLC25A45 protein_coding solute carrier Down-regulated in family 25, member the presence of 45 dyplasia ENSG00000205268 5150 PDE7A protein_coding phosphodiesterase Down-regulated in 7A the presence of dyplasia ENSG00000160712 3570 IL6R protein_coding interleukin 6 Down-regulated in receptor the presence of dyplasia ENSG00000119906 55719 FAM178A protein_coding family with Down-regulated in sequence similarity the presence of 178, member A dyplasia ENSG00000166762 117155 CATSPER2 protein_coding cation channel, Down-regulated in sperm associated 2 the presence of dyplasia ENSG00000203709 NA Clorf132 protein_coding Down-regulated in the presence of dyplasia ENSG00000167202 23102 TBC1D2B protein_coding TBC1 domain Down-regulated in family, member 2B the presence of dyplasia ENSG00000140326 146059 CDAN1 protein_coding congenital Down-regulated in dyserythropoietic the presence of anemia, type I dyplasia ENSG00000238105 55592 pseudogene golgin A2 Down-regulated in pseudogene 5 the presence of dyplasia ENSG00000167395 9726 ZNF646 protein_coding zinc finger protein Down-regulated in 646 the presence of dyplasia ENSG00000109063 4621 MYH3 protein_coding myosin, heavy Down-regulated in chain 3, skeletal the presence of muscle, embryonic dyplasia ENSG00000196689 7442 TRPV1 protein_coding transient receptor Down-regulated in potential cation the presence of channel, subfamily dyplasia V, member 1 ENSG00000168488 11273 ATXN2L protein_coding ataxin 2-like Down-regulated in the presence of dyplasia ENSG00000230124 100527964 antisense uncharacterized Down-regulated in LOC100527964 the presence of dyplasia ENSG00000184551 NA pseudogene Down-regulated in the presence of dyplasia ENSG00000198026 63925 ZNF335 protein_coding zinc finger protein Down-regulated in 335 the presence of dyplasia ENSG00000166887 23339 VPS39 protein_coding vacuolar protein Down-regulated in sorting 39 homolog the presence of (S. cerevisiae) dyplasia ENSG00000006530 55750 AGK protein_coding acylglycerol kinase Down-regulated in the presence of dyplasia ENSG00000128191 100302197 DGCR8 protein_coding DiGeorge Down-regulated in syndrome critical the presence of region gene 8 dyplasia ENSG00000109118 57649 PHF12 protein_coding PHD finger protein Down-regulated in 12 the presence of dyplasia ENSG00000068400 56850 GRIPAP1 protein_coding GRIP1 associated Down-regulated in protein 1 the presence of dyplasia ENSG00000228544 100131193 antisense uncharacterized Down-regulated in LOC100131193 the presence of dyplasia ENSG00000204842 6311 ATXN2 protein_coding ataxin 2 Down-regulated in the presence of dyplasia ENSG00000084774 790 CAD protein_coding carbamoyl- Down-regulated in phosphate the presence of synthetase 2, dyplasia aspartate transcarbamylase, and dihydroorotase ENSG00000184787 7327 UBE2G2 protein_coding ubiquitin- Down-regulated in conjugating the presence of enzyme E2G 2 dyplasia ENSG00000173120 22992 KDM2A protein_coding lysine (K)-specific Down-regulated in demethylase 2A the presence of dyplasia ENSG00000215012 79680 C22orf29 protein_coding chromosome 22 Down-regulated in open reading frame the presence of 29 dyplasia ENSG00000135365 51317 PHF21A protein_coding PHD finger protein Down-regulated in 21A the presence of dyplasia ENSG00000157827 114793 FMNL2 protein_coding formin-like 2 Down-regulated in the presence of dyplasia ENSG00000112659 23113 CUL9 protein_coding cullin 9 Down-regulated in the presence of dyplasia ENSG00000108509 23125 CAMTA2 protein_coding calmodulin binding Down-regulated in transcription the presence of activator 2 dyplasia ENSG00000170919 100190939 TPT1-AS1 lincRNA TPT1 antisense Down-regulated in RNA 1 (non- the presence of protein coding) dyplasia ENSG00000197622 56882 CDC42SE1 protein_coding CDC42 small Down-regulated in effector 1 the presence of dyplasia ENSG00000100888 57680 CHD8 protein_coding chromodomain Down-regulated in helicase DNA the presence of binding protein 8 dyplasia ENSG00000213983 8906 AP1G2 protein_coding adaptor-related Down-regulated in protein complex 1, the presence of gamma 2 subunit dyplasia ENSG00000130827 55558 PLXNA3 protein_coding plexin A3 Down-regulated in the presence of dyplasia ENSG00000198169 90987 ZNF251 protein_coding zinc finger protein Down-regulated in 251 the presence of dyplasia ENSG00000132424 25957 PNISR protein_coding PNN-interacting Down-regulated in serine/arginine-rich the presence of protein dyplasia ENSG00000120709 51307 FAM53C protein_coding family with Down-regulated in sequence similarity the presence of 53, member C dyplasia ENSG00000131067 2686 GGT7 protein_coding gamma- Down-regulated in glutamyltransferase the presence of 7 dyplasia ENSG00000166888 6778 STAT6 protein_coding signal transducer Down-regulated in and activator of the presence of transcription 6, dyplasia interleukin-4 induced ENSG00000258727 NA antisense Down-regulated in the presence of dyplasia ENSG00000141867 23476 BRD4 protein_coding bromodomain Down-regulated in containing 4 the presence of dyplasia ENSG00000005339 1387 CREBBP protein_coding CREB binding Down-regulated in protein the presence of dyplasia ENSG00000165275 158234 RG9MTD3 protein_coding RNA (guanine-9-) Down-regulated in methyltransferase the presence of domain containing dyplasia 3 ENSG00000196535 399687 MYO18A protein_coding myosin XVIIIA Down-regulated in the presence of dyplasia ENSG00000125814 63908 NAPB protein_coding N-ethylmaleimide- Down-regulated in sensitive factor the presence of attachment protein, dyplasia beta ENSG00000092421 57556 SEMA6A protein_coding sema domain, Down-regulated in transmembrane the presence of domain (TM), and dyplasia cytoplasmic domain, (semaphorin) 6A ENSG00000137497 4926 NUMA1 protein_coding nuclear mitotic Down-regulated in apparatus protein 1 the presence of dyplasia ENSG00000100416 55687 TRMU protein_coding tRNA 5- Down-regulated in methylaminomethy the presence of 1-2-thiouridylate dyplasia methyltransferase ENSG00000110274 22897 CEP164 protein_coding centrosomal protein Down-regulated in 164 kDa the presence of dyplasia ENSG00000104885 84444 DOT1L protein_coding DOT1-like, histone Down-regulated in H3 the presence of methyltransferase dyplasia (S. cerevisiae) ENSG00000244161 100506906 FLNB- antisense FLNB antisense Down-regulated in AS1 RNA 1 (non- the presence of protein coding) dyplasia ENSG00000218418 NA pseudogene Down-regulated in the presence of dyplasia ENSG00000171163 55657 ZNF692 protein_coding zinc finger protein Down-regulated in 692 the presence of dyplasia ENSG00000184313 374977 HEATR8 protein_coding HEAT repeat Down-regulated in containing 8 the presence of dyplasia ENSG00000156858 78994 PRR14 protein_coding proline rich 14 Down-regulated in the presence of dyplasia ENSG00000247743 NA NA NA NA Down-regulated in the presence of dyplasia ENSG00000213015 51157 ZNF580 protein_coding zinc finger protein Down-regulated in 580 the presence of dyplasia ENSG00000142937 94163 RPS8 protein_coding ribosomal protein Up-regulated in the S8 presence of dyplasia ENSG00000129518 55837 EAPP protein_coding E2F-associated Up-regulated in the phosphoprotein presence of dyplasia ENSG00000213326 NA RPS7P11 pseudogene Up-regulated in the presence of dyplasia ENSG00000177889 7334 UBE2N protein_coding ubiquitin- Up-regulated in the conjugating presence of enzyme E2N dyplasia ENSG00000185834 NA RPL12P4 pseudogene Up-regulated in the presence of dyplasia ENSG00000166171 25911 DPCD protein_coding deleted in primary Up-regulated in the ciliary dyskinesia presence of homolog (mouse) dyplasia ENSG00000235297 NA pseudogene Up-regulated in the presence of dyplasia ENSG00000181163 4869 NPM1 protein_coding nucleophosmin Up-regulated in the (nucleolar presence of phosphoprotein dyplasia B23, numatrin) ENSG00000177600 619565 RPLP2 protein_coding ribosomal protein, Up-regulated in the large, P2 presence of dyplasia ENSG00000082515 29093 MRPL22 protein_coding mitochondrial Up-regulated in the ribosomal protein presence of L22 dyplasia ENSG00000185068 404672 GTF2H5 protein_coding general Up-regulated in the transcription factor presence of IIH, polypeptide 5 dyplasia ENSG00000134248 10542 HBXIP protein_coding hepatitis B virus x Up-regulated in the interacting protein presence of dyplasia ENSG00000186198 123264 protein_coding organic solute Up-regulated in the transporter beta presence of dyplasia ENSG00000186132 130355 C2orf76 protein_coding chromosome 2 Up-regulated in the open reading frame presence of 76 dyplasia ENSG00000185641 NA pseudogene Up-regulated in the presence of dyplasia ENSG00000168653 4725 NDUFS5 protein_coding NADH Up-regulated in the dehydrogenase presence of (ubiquinone) Fe-S dyplasia protein 5, 151 kDa (NADH-coenzyme Q reductase) ENSG00000100554 51382 ATP6V1D protein_coding ATPase, H+ Up-regulated in the transporting, presence of lysosomal 34 kDa, dyplasia V1 subunit D ENSG00000161016 6132 RPL8 protein_coding ribosomal protein Up-regulated in the L8 presence of dyplasia ENSG00000111775 1337 COX6A1 protein_coding cytochrome c Up-regulated in the oxidase subunit VIa presence of polypeptide 1 dyplasia ENSG00000183978 28958 CCDC56 protein_coding coiled-coil domain Up-regulated in the containing 56 presence of dyplasia ENSG00000236552 728658 RPL13AP5 pseudogene ribosomal protein Up-regulated in the L13a pseudogene 5 presence of dyplasia ENSG00000236801 NA pseudogene Up-regulated in the presence of dyplasia ENSG00000131100 529 ATP6V1E1 protein_coding ATPase, H+ Up-regulated in the transporting, presence of lysosomal 31 kDa, dyplasia V1 subunit E1 ENSG00000235174 NA RPL39P3 pseudogene Up-regulated in the presence of dyplasia ENSG00000169740 7580 ZNF32 protein_coding zinc finger protein Up-regulated in the 32 presence of dyplasia ENSG00000129562 1603 DAD1 protein_coding defender against Up-regulated in the cell death 1 presence of dyplasia ENSG00000144713 6161 RPL32 protein_coding ribosomal protein Up-regulated in the L32 presence of dyplasia ENSG00000197756 6168 RPL37A protein_coding ribosomal protein Up-regulated in the L37a presence of dyplasia ENSG00000164751 5828 PEX2 protein_coding peroxisomal Up-regulated in the biogenesis factor 2 presence of dyplasia ENSG00000010278 100652804 CD9 protein_coding CD9 molecule Up-regulated in the presence of dyplasia ENSG00000140988 26784 RPS2 protein_coding ribosomal protein Up-regulated in the S2 presence of dyplasia ENSG00000198618 NA PPIAP22 pseudogene Up-regulated in the presence of dyplasia ENSG00000151465 8872 CDC123 protein_coding cell division cycle Up-regulated in the 123 homolog (S. presence of cerevisiae) dyplasia ENSG00000143543 10899 JTB protein_coding jumping Up-regulated in the translocation presence of breakpoint dyplasia ENSG00000244398 NA pseudogene Up-regulated in the presence of dyplasia ENSG00000232856 NA protein_coding Up-regulated in the presence of dyplasia ENSG00000108100 219771 CCNY protein_coding cyclin Y Up-regulated in the presence of dyplasia ENSG00000118939 7347 UCHL3 protein_coding ubiquitin carboxyl- Up-regulated in the terminal esterase presence of L3 (ubiquitin dyplasia thiolesterase) ENSG00000169021 7386 UQCRFS1 protein_coding ubiquinol- Up-regulated in the cytochrome c presence of reductase, Rieske dyplasia iron-sulfur polypeptide 1 ENSG00000172809 6169 RPL38 protein_coding ribosomal protein Up-regulated in the L38 presence of dyplasia ENSG00000137154 6194 RPS6 protein_coding ribosomal protein Up-regulated in the S6 presence of dyplasia ENSG00000164405 27089 UQCRQ protein_coding ubiquinol- Up-regulated in the cytochrome c presence of reductase, complex dyplasia III subunit VII, 9.5 kDa ENSG00000143457 55204 GOLPH3L protein_coding golgi Up-regulated in the phosphoprotein 3- presence of like dyplasia ENSG00000138297 100287932 TIMM23 protein_coding translocase of inner Up-regulated in the mitochondrial presence of membrane 23 dyplasia homolog (yeast) ENSG00000228474 100128731 OST4 protein_coding oligosaccharyltrans Up-regulated in the ferase 4 homolog presence of (S. cerevisiae) dyplasia ENSG00000112981 8382 NME5 protein_coding non-metastatic cells Up-regulated in the 5, protein presence of expressed in dyplasia (nucleoside- diphosphate kinase) ENSG00000112667 10591 C6orf108 protein_coding chromosome 6 Up-regulated in the open reading frame presence of 108 dyplasia ENSG00000183617 116541 MRPL54 protein_coding mitochondrial Up-regulated in the ribosomal protein presence of L54 dyplasia ENSG00000188873 NA RPL10AP2 pseudogene Up-regulated in the presence of dyplasia ENSG00000131143 1327 COX411 protein_coding cytochrome c Up-regulated in the oxidase subunit IV presence of isoform 1 dyplasia ENSG00000178741 9377 COX5A protein_coding cytochrome c Up-regulated in the oxidase subunit Va presence of dyplasia ENSG00000232112 51372 CCDC72 protein_coding coiled-coil domain Up-regulated in the containing 72 presence of dyplasia ENSG00000178449 84987 COX14 protein_coding COX14 Up-regulated in the cytochrome c presence of oxidase assembly dyplasia homolog (S. cerevisiae) ENSG00000138663 51138 COPS4 protein_coding COP9 constitutive Up-regulated in the photomorphogenic presence of homolog subunit 4 dyplasia (Arabidopsis) ENSG00000149547 9538 E124 protein_coding etoposide induced Up-regulated in the 2.4 mRNA presence of dyplasia ENSG00000173660 440567 UQCRH protein_coding ubiquinol- Up-regulated in the cytochrome c presence of reductase hinge dyplasia protein ENSG00000125356 4694 NDUFA1 protein_coding NADH Up-regulated in the dehydrogenase presence of (ubiquinone) 1 dyplasia alpha subcomplex, 1, 7.5 kDa ENSG00000162244 6159 RPL29 protein_coding ribosomal protein Up-regulated in the L29 presence of dyplasia ENSG00000174444 595097 RPL4 protein_coding ribosomal protein Up-regulated in the L4 presence of dyplasia ENSG00000145247 132299 OCIAD2 protein_coding OCIA domain Up-regulated in the containing 2 presence of dyplasia ENSG00000178980 6415 SEPW1 protein_coding selenoprotein W, 1 Up-regulated in the presence of dyplasia ENSG00000169020 521 ATP5I protein_coding ATP synthase, H+ Up-regulated in the transporting, presence of mitochondrial Fo dyplasia complex, subunit E ENSG00000125743 6633 SNRPD2 protein_coding small nuclear Up-regulated in the ribonucleoprotein presence of D2 polypeptide dyplasia 16.5 kDa ENSG00000101928 56180 MOSPD1 protein_coding motile sperm Up-regulated in the domain containing presence of 1 dyplasia ENSG00000151366 100532726 NDUFC2 protein_coding NADH Up-regulated in the dehydrogenase presence of (ubiquinone) 1, dyplasia subcomplex unknown, 2, 14.5 kDa ENSG00000171421 64979 MRPL36 protein_coding mitochondrial Up-regulated in the ribosomal protein presence of L36 dyplasia ENSG00000198755 4736 RPL10A protein_coding ribosomal protein Up-regulated in the L10a presence of dyplasia ENSG00000232119 28985 MCTS1 protein_coding malignant T cell Up-regulated in the amplified sequence presence of 1 dyplasia ENSG00000198643 131177 FAM3D protein_coding family with Up-regulated in the sequence similarity presence of 3, member D dyplasia ENSG00000123144 79002 C19orf43 protein_coding chromosome 19 Up-regulated in the open reading frame presence of 43 dyplasia ENSG00000111669 7167 TPI1 protein_coding triosephosphate Up-regulated in the isomerase 1 presence of dyplasia ENSG00000089063 29058 TMEM230 protein_coding chromosome 20 Up-regulated in the open reading frame presence of 30 dyplasia ENSG00000214026 6150 MRPL23 protein_coding mitochondrial Up-regulated in the ribosomal protein presence of L23 dyplasia ENSG00000119421 4702 NDUFA8 protein_coding NADH Up-regulated in the dehydrogenase presence of (ubiquinone) 1 dyplasia alpha subcomplex, 8, 19 kDa ENSG00000135940 1329 COX5B protein_coding cytochrome c Up-regulated in the oxidase subunit Vb presence of dyplasia ENSG00000146066 192286 HIGD2A protein_coding HIG1 hypoxia Up-regulated in the inducible domain presence of family, member 2A dyplasia ENSG00000170892 79042 TSEN34 protein_coding tRNA splicing Up-regulated in the endonuclease 34 presence of homolog (S. dyplasia cerevisiae) ENSG00000166920 84419 C15orf48 protein_coding chromosome 15 Up-regulated in the open reading frame presence of 48 dyplasia ENSG00000140307 2958 GTF2A2 protein_coding general Up-regulated in the transcription factor presence of IIA, 2, 12 kDa dyplasia ENSG00000184831 79135 APOO protein_coding apolipoprotein O Up-regulated in the presence of dyplasia ENSG00000205544 254863 C17orf61 protein_coding chromosome 17 Up-regulated in the open reading frame presence of 61 dyplasia

Supplemental Table 1. ANOVA derived p-values for the association between the surrogate variables and demographic/phenotypic variables Variable SV1 SV2 SV3 SV4 SV5 SV6 SV7 SV8 SV9 Presence of 0.549 0.376 0.964 0.500 0.118 0.481 0.046 0.166 0.652 premalignant lesion (2-level) Smoking status 0.000 0.655 0.191 0.084 0.689 0.804 0.308 0.719 0.761 Smoking status by 0.000 0.363 0.801 0.045 0.819 0.780 0.130 0.827 0.663 Gene Expression Sex 0.961 0.058 0.000 0.032 0.492 0.801 0.433 0.884 0.991 COPD status 0.612 0.866 0.047 0.161 0.973 0.129 0.083 0.007 0.592 Pack-years 0.398 0.293 0.523 0.576 0.845 0.399 0.875 0.428 0.178 Age 0.300 0.153 0.562 0.845 0.166 0.618 0.037 0.050 0.528 FEV1 0.050 0.391 0.046 0.009 0.123 0.150 0.171 0.028 0.691 FEV1/FVC ratio 0.023 0.670 0.172 0.056 0.491 0.107 0.028 0.011 0.708 Barcode 0.870 0.605 0.006 0.500 0.745 0.444 0.695 0.119 0.187 Lane 0.335 0.748 0.682 0.351 0.037 0.792 0.402 0.996 0.549 Batch 0.676 0.730 0.474 0.426 0.861 0.037 0.145 0.688 0.261 GC content 0.599 0.886 0.057 0.902 0.257 0.157 0.001 0.416 0.210 Genebody 80/20 ratio 0.000 0.245 0.633 0.271 0.000 0.736 0.015 0.319 0.048 (gb-ratio) Number of Uniquely 0.302 0.154 0.726 0.948 0.055 0.120 0.036 0.163 0.586 Aligning Reads Number of Reads 0.545 0.605 0.498 0.442 0.000 0.383 0.170 0.745 0.942 Aligning to Splice Junctions Z-score (sample mean 0.514 0.371 0.238 0.595 0.024 0.031 0.005 0.353 0.021 of z-score normalized data by gene) Relative Expression 0.814 0.615 0.996 0.740 0.918 0.887 0.214 0.274 0.111 (sample median of ratios computed for each gene by dividing the expression by the median expression)

Supplemental Table 2. Phenotypic information about the human biopsy cell cultures used in the bioenergetics experiments. Smoking Bio- Mito- Histology Gender Status energetics TrackerFM Normal F Current X Normal M Current X Normal F Former X Normal M Former X Normal F Current X X Normal F Current X X Moderate M Current X Dysplasia Severe M Former X Dysplasia Severe M Current X Dysplasia Low grade M Former X dysplasia Severe M Current X X Dysplasia Low grade M Former X X dysplasia

Supplemental Table 3. Phenotypic information about the human biopsies used in the IHC experiments. Smoking Stain PtID Status WorstHistology_Description Tomm-22 Pt 3 FS 0 Normal, Negative, Benign Mucosa Cox-IV Pt 3 FS 0 Normal, Negative, Benign Mucosa Tomm-22 Pt 4 FS 23 Squamous Metaplasia (non-specific), Mature Metaplasia, Squamous Hyperplasia Cox-IV Pt 4 FS 23 Squamous Metaplasia (non-specific), Mature Metaplasia, Squamous Hyperplasia Tomm-22 Pt 3 FS 25 Moderate Dysplasia, Squamous Pre-invasive Cox-IV Pt 3 FS 25 Moderate Dysplasia, Squamous Pre-invasive Tomm-22 Pt 1 CS 27 CIS Squamous Carcinoma In-Situ Cox-IV Pt 1 CS 27 CIS Squamous Carcinoma In-Situ (*CS refers to current smoker and FS to former smoker)

Supplemental Table 4. Demographic and clinical characteristics of the British Columbia Lung Health Study stratified by premalignant lesions status Discovery Set Validation Set Overall No Lesions Lesions Overall No Lesions Lesions Factor (n = 58) (n = 20) (n = 38) P* (n = 17) (n = 5) (n = 12) P* Age 62.7 (7.1) 64.1 (5.8) 61.9 (7.6) 0.24 63.9 (8.6) 66 (5.8) 63 (9.7) 0.45 Male 37/58 (63.8) 12/20 (60) 25/38 (65.8) 0.78 14/17 (82.4) 4/5 (80) 10/12 (83.3) 1 Current smoker 28/58 (48.3) 9/20 (45) 19/38 (50) 0.79 8/17 (47.1) 2/5 (40) 6/12 (50) 1 Pack-years 48.2 (16.9) 49.4 (18.9) 47.5 (15.9) 0.71 44.6 (12.9) 40.5 (11.6) 46.3 (13.5) 0.39 FEV1% Predicted 86.5 (17.7) 87.8 (16.7) 85.7 (18.5) 0.66 69.5 (16.2) 71 (17.7) 68.9 (16.3) 0.83 FEV1/FVC Ratio 72.1 (7.7) 75.1 (6.3) 70.4 (8) 0.02 67 (8.1) 66.8 (8.5) 67.1 (8.3) 0.95 COPD (FEV1 % < 80 & 11/58 (19) 2/20 (10) 9/38 (23.7) 0.3 11/17 (64.7) 3/5 (60) 8/12 (66.7) 1 FEV1/FVC < 70) Histology <0.001 <0.001 Normal 11/58 (19) 11/20 (55) 1/17 (5.9) 1/5 (20) Hyperplasia 9/58 (15.5) 9/20 (45) 4/17 (23.5) 4/5 (80) Metaplasia 0/58 (0) 0/17 (0) Mild Dysplasia 29/58 (50) 29/38 (76.3) 6/17 (35.3) 6/12 (50) Moderate Dysplasia 6/58 (10.3) 6/38 (15.8) 6/17 (35.3) 6/12 (50) Severe Dysplasia 3/58 (5.2) 3/38 (7.9) 0/12 (0) Data are means (SD) for continuous variables and proportions (%) dichotomous variables. Reads are expressed in millions denoted by M. P* values are for the comparison of subjects with and without premalignant lesions. Two sample t-tests were used for continuous variables; Fisher's exact test was used for factors.

Supplemental Table 5. Alignment statistics of the British Columbia Lung Health Study Discovery and the Roswell Park Cancer Institute cohort BC-LHS Discovery Set BC-LHS Validation Set RPCI Overall No Lesions Lesions Overall No Lesions Lesions Overall Factor (n = 58) (n = 20) (n = 38) P* (n = 17) (n = 5) (n = 12) P* (n = 51) Total Alignments 90M (16M) 98M (15M) 91M (17M) 0.67 93M (22M) 94M (18M) 92M (24M) 0.86 95M (15M) Unique Alignments 83M (15M) 82M (13M) 83M (16M) 0.65 85M (20M) 86M (16M) 84M (22M) 0.85 Properly Paired Alignments 66M (1.2M) 65M (11M) 67M (12M) 0.63 68M (16M) 69M (13M) 67M (17M) 0.86 65M (9.6M) Genebody 80/20 Ratio 1.3 (0.2) 1.3 (0.1) 1.3 (0.2) 0.39 1.3 (0.3) 1.2 (0.1) 1.4 (0.3) 0.15 1.8 (0.2) Mean GC Content 48.1 (3.4) 47.5 (2.7) 48.4 (3.6) 0.33 47.4 (3.8) 46.9 (3.8) 47.6 (3.9) 0.74 49.2 (1.4) Data are means (SD). Reads are expressed in millions denoted by M. P* values are for two sample t-tests for comparison of subjects with and without premalignant lesions.

Supplemental Table 6. Demographic and clinical characteristics of the Roswell Park Cancer Institute Cohort (n = 51 samples from n = 23 subjects) Progressing Factor Overall Regressing Stable P* No. Samples 51 34 22 No. Sample 28 17 11 Pairs No. Patients** 23 16 10 Time between 343.8 (171.9) 350.9 (199.6)  332.8 (125.9) 0.77 Procedures (Days) Histological −0.9 (1.7)  −1.9 (1.0)   0.7 (1.3) <0.001 Grade Change Worst Histological Lesion Observed Normal 5/51 (9.8)  4/34 (11.8) 2/22 (9.1) 0.038 Hyperplasia 6/51 (11.8) 5/34 (14.7) 1/22 (4.5) Metaplasia 9/51 (17.6) 8/34 (23.5) 1/22 (4.5) Mild Dysplasia 3/51 (5.9)  3/34 (8.8)   0 (0) Moderate 20/51 (39.2)  9/34 (26.5) 15/22 (68.2) Dysplasia Severe 8/51 (15.7) 5/34 (14.7)  3/22 (13.6) Dysplasia Age at 58.1 (6.5)  58.4 (6.9)  57.6 (6.1) 1 Baseline Male 13/28 (46.4)  7/17 (41.2)  6/11 (54.5) 0.7 Ever smoker at 27/28 (96.4)  17/17 (100)   10/11 (90.9) 0.39 Baseline Pack-years at 48.1 (22)   49.8 (24.8)  45.4 (17.6) 1 Baseline Data are means (SD) for continuous variables and proportions (%) for dichotomous variables. P*values are for the comparison of samples, sample pairs, or patients classified as having regressing or progressing/stable PMLs. Two sample t-tests were used for continuous variables; Fisher's exact test was used for factors. **Among the 23 patients, 3 patients had 2 sample pairs where one pair was classified as regressing and the other as progressing/stable. These patients are counted in both the regressing and progressing/stable columns.

Dataset 1. Ensembl IDs for genes used to predict smoking status. ENSG00000151632 ENSG00000125398 ENSG00000159228 ENSG00000109586 ENSG00000049089 ENSG00000198431 ENSG00000140961 ENSG00000117450 ENSG00000111058 ENSG00000198074 ENSG00000001084 ENSG00000168309 ENSG00000108602 ENSG00000065833 ENSG00000215182 ENSG00000079819 ENSG00000117983 ENSG00000163931 ENSG00000173376 ENSG00000197838 ENSG00000176153 ENSG00000136810 ENSG00000137642 ENSG00000134873 ENSG00000172765 ENSG00000154040 ENSG00000048707 ENSG00000123124 ENSG00000102359 ENSG00000197747 ENSG00000103222 ENSG00000103647 ENSG00000099968 ENSG00000196344 ENSG00000140939 ENSG00000167996 ENSG00000006125 ENSG00000149256 ENSG00000010404 ENSG00000023909 ENSG00000077147 ENSG00000134775 ENSG00000177156 ENSG00000123700 ENSG00000124664 ENSG00000197712 ENSG00000154822 ENSG00000086548 ENSG00000137573 ENSG00000100012 ENSG00000136205 ENSG00000138061 ENSG00000104341 ENSG00000151012 ENSG00000039537 ENSG00000181458 ENSG00000006210 ENSG00000078596 ENSG00000117394 ENSG00000106541 ENSG00000125798 ENSG00000109854 ENSG00000196139 ENSG00000162496 ENSG00000181019 ENSG00000140526 ENSG00000166670 ENSG00000198417 ENSG00000162804 ENSG00000105388 ENSG00000069764 ENSG00000108924 ENSG00000171903 ENSG00000085662 ENSG00000137648 ENSG00000125144 ENSG00000113924 ENSG00000134827 ENSG00000142655 ENSG00000139629 ENSG00000160180 ENSG00000124107 ENSG00000119514 ENSG00000227051 ENSG00000144711 ENSG00000101445 ENSG00000137337 ENSG00000114638 ENSG00000142657 ENSG00000130595 ENSG00000145147 ENSG00000087842 ENSG00000133985 ENSG00000125813

Dataset 2. Results of pathway enrichment using ROAST (FDR < 0.05). The column “Direction” refers to pathway enrichment among genes up-regulated (Up) or down-regulated (Down) in the presence of PMLs. Pathway NGenes PropDown PropUp Direction PValue FDR REACTOME_METABOLISM_OF_PROTEINS 382 0.091623 0.544503 Up 0.002 0.0128 REACTOME_METABOLISM_OF_RNA 251 0.139442 0.494024 Up 0.002 0.0128 REACTOME_METABOLISM_OF_MRNA 206 0.131068 0.533981 Up 0.002 0.0128 KEGG_HUNTINGTONS_DISEASE 158 0.126582 0.607595 Up 0.002 0.0128 KEGG_ALZTIEIMERS_DISEASE 141 0.120567 0.631206 Up 0.002 0.0128 REACTOME_TRANSLATION 141 0.042553 0.780142 Up 0.002 0.0128 REACTOME_INFLUENZA_LIFE_CYCLE 133 0.075188 0.691729 Up 0.002 0.0128 REACTOME_TCA_CYCLE_AND_RESPIRATORY_ELECTRON_ 125 0.088 0.64 Up 0.002 0.0128 TRANSPORT KEGG_OXIDATIVE_PHOSPHORYLATION 117 0.042735 0.692308 Up 0.002 0.0128 KEGG_PARKINSONS_DISEASE 113 0.079646 0.699115 Up 0.002 0.0128 REACTOME_SRP_DEPENDENT_COTRANSLATIONAL_ 105 0.019048 0.885714 Up 0.002 0.0128 PROTEIN_TARGETING_TO_MEMBRANE REACTOME_NONSENSE_MEDIATED_DECAY_ 103 0.07767 0.776699 Up 0.002 0.0128 ENHANCED_BY_THE_EXON_JUNCTION_COMPLEX REACTOME_3_UTR_MEDIATED_TRANSLATIONAL_ 102 0.029412 0.843137 Up 0.002 0.0128 REGULATION REACTOME_SIGNALING_BY_RHO_GTPASES 93 0.387097 0.150538 Down 0.002 0.0128 REACTOME_RESPIRATORY_ELECTRON_TRANSPORT_ 91 0.021978 0.758242 Up 0.002 0.0128 ATP_SYNTHESIS_BY_CHEMIOSMOTIC_CO

AT_PRODUCTION_BY_UNCOUPLING_PROTEINS_KEGG_ JAK_STAT_SIGNALING_PATHWAY 87 0.321839 0.126437 Down 0.002 0.0128 KEGG_PYRIMIDINE_METABOLISM 84 0.154762 0.380952 Up 0.002 0.0128 KEGG_RIBOSOME 83 0.012048 0.939759 Up 0.002 0.0128 REACTOME_PEPTIDE_CHAIN_ELONGATION 82 0.012195 0.939024 Up 0.002 0.0128 REACTOME_RESPIRATORY_ELECTRON_TRANSPORT 74 0.013514 0.756757 Up 0.002 0.0128 PID_HDAC_CLASSI_PATHWAY 60 0.366667 0.15 Down 0.002 0.0128 PID_MYC_REPRESSPATHWAY 55 0.381818 0.127273 Down 0.002 0.0128 REACTOME_ACTIVATION_OF_THE_MRNA_UPON_ 55 0.054545 0.745455 Up 0.002 0.0128 BINDING_OF_THE_CAP_BINDING_COMPLEX_A

_ SUBSEQUENT_BINDING_TO_43S PID_AVB3_INTEGRIN_PATHWAY 53 0.320755 0.132075 Down 0.002 0.0128 KEGG_ADIPOCYTOKINE_SIGNALING_PATHWAY 51 0.411765 0.176471 Down 0.002 0.0128 REACTOME_MITOCHONDRIAL_PROTEIN_IMPORT 49 0.102041 0.530612 Up 0.002 0.0128 REACTOME_FORMATION_OF_THE_TERNARY_ 47 0.042553 0.829787 Up 0.002 0.0128 COMPLEX_AND_SUBSEQUENTLY_THE_43S_COMP

KEGG_CARDIAC_MUSCLE_CONTRACTION 43 0.116279 0.55814 Up 0.002 0.0128 KEGG_LYSINE_DEGRADATION 42 0.428571 0.166667 Down 0.002 0.0128 PID_IL4_2PATHWAY 42 0.380952 0.119048 Down 0.002 0.0128 REACTOME_FORMATION_OF_RNA_POL_II_ 41 0.170732 0.439024 Up 0.002 0.0128 ELONGATION_COMPLEX_ KEGG_NOTCH_SIGNALING_PATHWAY 40 0.425 0.125 Down 0.002 0.0128 PID_RHOA_REG_PATHWAY 40 0.475 0.125 Down 0.002 0.0128 REACTOME_NRAGE_SIGNALS_DEATH_THROUGH_INK 39 0.358974 0.153846 Down 0.002 0.0128 REACTOME_PRE_NOTCH_EXPRESSION_AND_ 38 0.342105 0.131579 Down 0.002 0.0128 PROCESSING REACTOME_NCAM_SIGNALING_FOR_NEURITE_OUT_ 37 0.459459 0.108108 Down 0.002 0.0128 GROWTH ST_GA13_PATHWAY 33 0.424242 0.121212 Down 0.002 0.0128 PID_RAC1_REG_PATHWAY 33 0.454545 0.121212 Down 0.002 0.0128 REACTOME_BMAL1_CLOCK_NPAS2_ACTIVATES_ 33 0.484848 0.090909 Down 0.002 0.0128 CIRCADIAN_EXPRESSION BIOCARTA_CARM_ER_PATHWAY 32 0.34375 0.125 Down 0.002 0.0128 REACTOME_G1_PHASE 32 0.09375 0.5 Up 0.002 0.0128 REACTOME_FORMATION_OF_THE_HIV1_EARLY_ 31 0.129032 0.483871 Up 0.002 0.0128 ELONGATION_COMPLEX KEGG_PROPANOATE_METABOLISM 30 0.1 0.433333 Up 0.002 0.0128 PID_FRA_PATHWAY 28 0.428571 0.071429 Down 0.002 0.0128 REACTOME_PURINE_METABOLISM 28 0.178571 0.392857 Up 0.002 0.0128 KEGG_BUTANOATE_METABOLISM 27 0.037037 0.481481 Up 0.002 0.0128 BIOCARTA_MYOSIN_PATHWAY 27 0.296296 0.111111 Down 0.002 0.0128 REACTOME_MRNA_CAPPING 27 0.111111 0.481481 Up 0.002 0.0128 REACTOME_FORMATION_OF_TRANSCRIPTION_ 27 0.074074 0.518519 Up 0.002 0.0128 COUPLED_NER_TC_NER_REPAIR_COMPLEX REACTOME_PRE_NOTCH_TRANSCRIPTION_AND_ 25 0.48 0.12 Down 0.002 0.0128 TRANSLATION ST_GAQ_PATHWAY 24 0.5 0.166667 Down 0.002 0.0128 REACTOME_RORA_ACTIVATES_CIRCADIAN_EXPRESSION 24 0.5 0.041667 Down 0.002 0.0128 REACTOME_ENDOSOMAL_SORTING_COMPLEX_ 24 0.083333 0.541667 Up 0.002 0.0128 REQUIRED_FOR_TRANSPORT_ESCRT BIOCARTA_HDAC_PATHWAY 23 0.478261 0.130435 Down 0.002 0.0128 PID_HDAC_CLASSIII_PATHWAY 22 0.454545 0.136364 Down 0.002 0.0128 PID_RXR_VDR_PATHWAY 22 0.409091 0.045455 Down 0.002 0.0128 REACTOME_PREFOLDIN_MEDIATED_TRANSFER_OF_ 21 0.047619 0.571429 Up 0.002 0.0128 SUBSTRATE_TO_CCT_TRIC REACTOME_SIGNALING_BY_FGFR1_MUTANTS 19 0.421053 0.157895 Down 0.002 0.0128 REACTOME_SIGNALING_BY_FGFR1_FUSION_MUTANTS 18 0.444444 0.111111 Down 0.002 0.0128 BIOCARTA_TNER2_PATHWAY 17 0.529412 0.117647 Down 0.002 0.0128 BIOCARTA_RELA_PATHWAY 15 0.533333 0.2 Down 0.002 0.0128 REACTOME_FORMATION_OF_ATP_BY_CHEMIOSMOTIC_ 15 0 0.866667 Up 0.002 0.0128 COUPLING REACTOME_EARLY_PHASE_OF_HIV_LIFE_CYCLE 13 0 0.538462 Up 0.002 0.0128 BIOCARTA_VDR_PATHWAY 12 0.583333 0 Down 0.002 0.0128 BIOCARTA_CARM1_PATHWAY 12 0.416667 0.166667 Down 0.002 0.0128 REACTOME_SEMA3A_PLEXIN_REPULSION_ 12 0.5 0.166667 Down 0.002 0.0128 SIGNALING_BY_INHIBITING_INTEGRIN_ADHESION BIOCARTA_ETC_PATHWAY 11 0 0.727273 Up 0.002 0.0128 BIOCARTA_EGER_SMRTE_PATHWAY 11 0.454545 0 Down 0.002 0.0128 BIOCARTA_P27_PATHWAY 11 0.090909 0.454545 Up 0.002 0.0128 PID_LPA4_PATHWAY 11 0.545455 0 Down 0.002 0.0128 REACTOME_PURINE_SALVAGE 11 0.181818 0.727273 Up 0.002 0.0128 BIOCARTA_RAB_PATHWAY 10 0 0.9 Up 0.002 0.0128 REACTOME_ASSOCIATION_OF_LICENSING_FACTORS_ 9 0.111111 0.555556 Up 0.002 0.0128 WITH_THE_PRE_REPLICATIVE_COMPLEX REACTOME_GLUTAMATE_NEUROTRANSMITTER_ 9 0.555556 0 Down 0.002 0.0128 RELEASE_CYCLE REACTOME_INTEGRATION_OF_PROVIRUS 8 0 0.625 Up 0.002 0.0128 BIOCARTA_NUCLEARRS_PATHWAY 6 0.5 0 Down 0.002 0.0128 REACTOME_ACYL_CHAIN_REMODELLING_OF_PI 6 0 0.666667 Up 0.002 0.0128 REACTOME_ENDOGENOUS_STEROLS 6 0.5 0.166667 Down 0.002 0.0128 REACTOME_SYNTHESIS_SECRETION_AND_ 6 0 0.833333 Up 0.002 0.0128 DEACYLATION_OF_GHRELIN REACTOME_INTERACTION_BETWEEN_L1_AND_ANKYRINS 6 1 0 Down 0.002 0.0128 KEGG_TAURINE_AND_HYPOTAURINE_METABOLISM 5 0.4 0.2 Down 0.002 0.0128 REACTOME_DOPAMINE_NEUROTRANSMITTER_ 5 0.6 0.2 Down 0.002 0.0128 RELEASE_CYCLE REACTOME_ACETYLCHOLINE_NEUROTRANSMITTER_ 4 0.75 0 Down 0.002 0.0128 RELEASE_CYCLE REACTOME_NUCLEAR_RECEPTOR_TRANSCRIPTION_ 34 0.294118 0.058824 Down 0.002 0.0128 PATHWAY KEGG_PROTEIN_EXPORT 23 0.043478 0.652174 Up 0.002 0.0128 ST_INTERLEUKIN_4_PATHWAY 23 0.391304 0.086957 Down 0.002 0.0128 REACTOME_TRAF6_MEDIATED_IRF7_ACTIVATION 17 0.529412 0 Down 0.002 0.0128 PID_CIRCADIANPATHWAY 15 0.533333 0.066667 Down 0.002 0.0128 REACTOME_VIRAL_MESSENGER_RNA_SYNTHESIS 14 0.071429 0.642857 Up 0.002 0.0128 REACTOME_METABOLISM_OF_POLYAMINES 13 0.076923 0.538462 Up 0.002 0.0128 REACTOME_NOTCH_HLH_TRANSCRIPTION_PATHWAY 11 0.454545 0.090909 Down 0.002 0.0128 REACTOME_ADENYLATE_CYCLASE_ACTIVATING_ 7 0.571429 0 Down 0.002 0.0128 PATHWAY ST_STAT3_PATHWAY 9 0.555556 0 Down 0.002 0.0128 REACTOME_BINDING_AND_ENTRY_OF_HIV_VIRION 4 0 0.5 Up 0.002 0.0128 PID_CD40_PATHWAY 27 0.333333 0.037037 Down 0.002 0.0128 REACTOME_CD28_DEPENDENT_PI3K_AKT_SIGNALING 19 0.473684 0.052632 Down 0.002 0.0128 BIOCARTA_RARRXR_PATHWAY 15 0.4 0.066667 Down 0.002 0.0128 BIOCARTA_PITX2_PATHWAY 13 0.384615 0 Down 0.002 0.0128 REACTOME_INCRETIN_SYNTHESIS_SECRETION_AND_ 9 0 0.444444 Up 0.002 0.0128 INACTIVATION REACTOME_CLASS_C_3_METABOTROPIC_GLUTAMATE_ 2 0.5 0 Down 0.002 0.0128 PHEROMONE_RECEPTORS BIOCARTA_EGF_PATHWAY 31 0.258065 0.032258 Down 0.002 0.0128 REACTOME_HDL_MEDIATED_LIPID_TRANSPORT 11 0.454545 0 Down 0.002 0.0128 REACTOME_GENERIC_TRANSCRIPTION_PATHWAY 292 0.349315 0.10274 Down 0.004 0.0283 REACTOME_DEVELOPMENTAL_BIOLOGY 270 0.333333 0.188889 Down 0.004 0.0283 REACTOME_SIGNALING_BY_PDGF 94 0.361702 0.148936 Down 0.004 0.0283 PID_SMAD2_3NUCLEARPATHWAY 68 0.411765 0.102941 Down 0.004 0.0283 PID_REG_GR_PATHWAY 60 0.366667 0.15 Down 0.004 0.0283 KEGG_ECM_RECEPTOR_INTERACTION 51 0.352941 0.117647 Down 0.004 0.0283 REACTOME_CIRCADIAN_CLOCK 48 0.416667 0.125 Down 0.004 0.0283 KEGG_PPAR_SIGNALING_PATHWAY 43 0.348837 0.162791 Down 0.004 0.0283 SIG_BCR_SIGNALING_PATHWAY 41 0.317073 0.04878 Down 0.004 0.0283 REACTOME_TRANSCRIPTION_COUPLED_NER_TC_NER 41 0.097561 0.439024 Up 0.004 0.0283 REACTOME_RNA_POL_II_TRANSCRIPTION_PRE_ 38 0.131579 0.447368 Up 0.004 0.0283 INITIATION_AND_PROMOTER_OPENING KEGG_AMYOTROPHIC_LATERAL_SCLEROSIS_ALS 37 0.189189 0.324324 Up 0.004 0.0283 KEGG_ABC_TRANSPORTERS 31 0.516129 0.129032 Down 0.004 0.0283 BIOCARTA_PAR1_PATHWAY 31 0.290323 0.16129 Down 0.004 0.0283 REACTOME_COLLAGEN_FORMATION 31 0.451613 0.096774 Down 0.004 0.0283 PID RETINOIC_ACID_PATHWAY 28 0.392857 0.178571 Down 0.004 0.0283 REACTOME_CIRCADIAN_REPRESSION_OF_EXPRESSION_ 22 0.5 0.045455 Down 0.004 0.0283 BY_REV_ERBA KEGG_O_GLYCAN_BIOSYNTHESIS 21 0.047619 0.619048 Up 0.004 0.0283 REACTOME_YAP1_AND_WWTR1_TAZ_STIMULATED_ 20 0.4 0.1 Down 0.004 0.0283 GENE_EXPRESSION BIOCARTA_AKT_PATHWAY 18 0.444444 0.166667 Down 0.004 0.0283 BIOCARTA_IL7_PATHWAY 16 0.4375 0.125 Down 0.004 0.0283 REACTOME_OXYGEN_DEPENDENT_PROLINE_ 15 0.066667 0.533333 Up 0.004 0.0283 HYDROXYLATION_OF_HYPOXIA_INDUCIBLE_FA BIOCARTA_IL22BP_PATHWAY 14 0.5 0 Down 0.004 0.0283 REACTOME_NCAM1_INTERACTIONS 14 0.571429 0 Down 0.004 0.0283 REACTOME_EFFECTS_OF_PIP2_HYDROLYSIS 14 0.428571 0.071429 Down 0.004 0.0283 KEGG_RIBOFLAVIN_METABOLISM 13 0.076923 0.461538 Up 0.004 0.0283 REACTOME_TRAF3_DEPENDENT_IRF_ACTIVATION_ 13 0.461538 0 Down 0.004 0.0283 PATHWAY BIOCARTA_EPONFKB_PATHWAY 9 0.666667 0 Down 0.004 0.0283 REACTOME_IL_6_SIGNALING 9 0.444444 0 Down 0.004 0.0283 REACTOME_SYNTHESIS_SECRETION_AND_ 7 0 0.571429 Up 0.004 0.0283 INACTIVATION_OF_GIP BIOCARTA_GABA_PATHWAY 3 0 0.666667 Up 0.004 0.0283 REACTOME_INFLUENZA_VIRAL_RNA_TRANSCRIPTION_ 98 0.020408 0.867347 Up 0.004 0.0283 AND_REPLICATION REACTOME_LIPOPROTEIN_METABOLISM 19 0.315789 0.052632 Down 0.004 0.0283 REACTOME_ACYL_CHAIN_REMODELLING_OF_PG 7 0 0.571429 Up 0.004 0.0283 BIOCARTA_PDGF_PATHWAY 30 0.266667 0.033333 Down 0.004 0.0283 REACTOME_SYNTHESIS_SECRETION_AND_ 8 0 0.5 Up 0.004 0.0283 INACTIVATION_OF_GLP1 BIOCARTA_SALMONELLA_PATHWAY 11 0 0.636364 Up 0.004 0.0283 REACTOME_AXON_GUIDANCE 173 0.34104 0.179191 Down 0.006 0.0386 REACTOME_SIGNALING_BY_NOTCH 90 0.311111 0.2 Down 0.006 0.0386 KEGG_PEROXISOME 71 0.098592 0.352113 Up 0.006 0.0386 ST_INTEGRIN_SIGNALING_PATHWAY 71 0.323944 0.126761 Down 0.006 0.0386 REACTOME_SEMAPHORIN_INTERACTIONS 58 0.310345 0.224138 Down 0.006 0.0386 REACTOME_RNA_POL_II_PRE_TRANSCRIPTION_EVENTS 57 0.175439 0.385965 Up 0.006 0.0386 KEGG_ACUTE_MYELOID_LEUKEMIA 53 0.320755 0.132075 Down 0.006 0.0386 REACTOME_NUCLEOTIDE_EXCISION_REPAIR 46 0.108696 0.391304 Up 0.006 0.0386 REACTOME_EXTRACELLULAR_MATRIX_ORGANIZATION 43 0.372093 0.093023 Down 0.006 0.0386 KEGG_VALINE_LEUCINE_AND_ISOLEUCINE_ 40 0.075 0.45 Up 0.006 0.0386 DEGRADATION PID_HDAC_CLASII_PATHWAY 31 0.419355 0.16129 Down 0.006 0.0386 REACTOME_ELONGATION_ARREST_AND_RECOVERY 31 0.193548 0.451613 Up 0.006 0.0386 KEGG_RNA_POLYMERASE 27 0.074074 0.481481 Up 0.006 0.0386 SIG_IL4RECEPTOR_IN_B_LYPHOCYTES 25 0.32 0.04 Down 0.006 0.0386 PID_REELINPATHWAY 24 0.416667 0.166667 Down 0.006 0.0386 REACTOME_ABC_FAMILY_PROTEINS_MEDIATED_ 23 0.521739 0.217391 Down 0.006 0.0386 TRANSPORT REACTOME_ABORTIVE_ELONGATION_OF_HIV1_ 23 0.130435 0.478261 Up 0.006 0.0386 TRANSCRIPT_IN_THE_ABSENCE_OF_TAT BIOCARTA_GH_PATHWAY 22 0.363636 0.045455 Down 0.006 0.0386 REACTOME_RNA_POL_III_CHAIN_ELONGATION 16 0.0625 0.4375 Up 0.006 0.0386 BIOCARTA_CD40_PATHWAY 14 0.5 0.071429 Down 0.006 0.0386 REACTOME_ACYL_CHAIN_REMODELLING_OF_PC 12 0.166667 0.5 Up 0.006 0.0386 REACTOME_CASPASE_MEDIATED_CLEAVAGE_OF_ 11 0.545455 0.272727 Down 0.006 0.0386 CYTOSKELETAL_PROTEINS REACTOME_ORGANIC_CATION_ANION_ZWITTERION_ 5 0.6 0 Down 0.006 0.0386 TRANSPORT KEGG_FOCAL_ADHESION 145 0.296552 0.151724 Down 0.006 0.0386 PID_TNFPATHWAY 43 0.395349 0.093023 Down 0.006 0.0386 REACTOME_APC_CDC20_MEDIATED_DEGRADATION_OF_ 18 0.111111 0.388889 Up 0.006 0.0386 NEK2A BIOCARTA_ETS_PATHWAY 17 0.352941 0.117647 Down 0.006 0.0386 PID_HIF1APATHWAY 18 0.166667 0.333333 Up 0.006 0.0386 KEGG_TRYPTOPHAN_METABOLISM 25 0.08 0.28 Up 0.006 0.0386 REACTOME_N_GLYCAN_ANTENNAE_ELONGATION 10 0.1 0.5 Up 0.006 0.0386 REACTOME_AMINO_ACID_TRANSPORT_ACROSS_THE_ 18 0.388889 0 Down 0.006 0.0386 PLASMA_MEMBRANE

indicates data missing or illegible when filed

Dataset 3. GSEA results detailing lung cancer associated dataset enrichment among genes differentially expressed in the airway field associated with PMLs NOM FDR FWER RANK Gene Set SIZE ES NES p-val q-val p-val AT MAX LEADING EDGE OOI ET AL. EARLY, DN-REG, 26 −0.56 −1.87 0.002 0.005 0.017 2634 tags = 46%, list = 19%, signal = 57% PVN P < 0.05, TVN P < 0.05 OOI ET AL. EARLY, UP-REG, 487 0.36 2.11 0 0 0.001 3850 tags = 43%, list = 28%, signal = 58% PVN P < 0.05, TVN P < 0.05 OOI ET AL. STEPWISE, DN-REG, 111 −0.31 −1.4 0.028 0.064 0.794 3041 tags = 27%, list = 22%, signal = 54% PVN P < 0.05, TVP P < 0.05, TVN P < 0.05 OOI ET AL. STEPWISE, UP-REG, 518 0.29 1.73 0. 0.005 0.076 2858 tags = 29%, list = 21%, signal = 35% PVN P < 0.05, TVP P < 0.05, TVN P < 0.05 OOI ET AL. LATE, DN-REG, 12 −0.64 −1.74 0.012 0.009 0.082 1784 tags = 58%, list = 13%, signal = 67% TVP P < 0.05, TVN P < 0.05 OOI ET AL. LATE, UP-REG, 54 0.53 2.24 0 0 0 3052 tags = 46%, list = 22%, signal = 59 TVP P < 0.05, TVN P < 0.05 TCGA, SCCVN, DN-REG, 200 119 −0.37 −1.67 0.001 0.014 0.152 3526 tags = 36%, list = 25%, signal = 48% TCGA, SCCVN, UP-REG, 200 146 0.28 1.41 0.013 0.048 0.6 3950 tags = 40%, list = 28%, signal = 55% GSE18842, TVN, DN-REG, 200 111 −0.42 −1.87 0 0.007 0.016 3526 tags = 41%, list = 25%, signal = 54% GSE18842, TVN, UP-REG, 200 149 0.43 2.14 0 0 0.001 4601 tags = 52%, list = 33%, signal = 77% GSE19188, SCCVN, DN-REG, 200 115 −0.35 −1.55 0.006 0.027 0.371 4837 tags = 50%, list = 35%, signal = 75% GSE19188, SCCVN, UP-REG, 200 147 0.42 2.14 0 0 0.001 3596 tags = 41%, list = 26%, signal = 55% GSE4115, CAVN, DN-REG, 200 108 −0.35 −1.56 0.005 0.031 0.365 3066 tags = 31%, list = 22%, signal = 39% GSE4115, CAVN, UP-REG, 200 197 0.45 2.36 0 0 0 3781 tags = 55%, list = 27%, signal = 74%

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1.-18. (canceled)
 19. A method of processing a sample from a subject suspected of having a premalignant bronchial lesion comprising the steps of: (a) providing a biological sample from the mouth or nose of the subject or from a brushing of the bronchi walls of the subject; and (b) measuring the expression of two or more genes in the sample by northern-blot hybridization, a ribonuclease protection assay, or a reverse transcriptase polymerase chain reaction (RT-PCR) method, wherein the two or more genes are genes involved in an oxidative phosphorylation (OXPHOS), electron transport chain (ETC), or mitochondrial protein transport pathway.
 20. The method of claim 19, wherein the expression of at least five genes involved in an oxidative phosphorylation (OXPHOS), electron transport chain (ETC), or mitochondrial protein transport pathway are measured.
 21. The method of claim 19, wherein the expression in the sample of at least twenty genes are measured.
 22. The method of claim 19, wherein the two or more genes comprise cDNA.
 23. The method of claim 19, wherein the expression of two or more genes in the sample is measured by an RT-PCR method.
 24. The method of claim 19, wherein the biological sample is obtained from the mouth of the subject.
 25. The method of claim 19, wherein the subject has a positive result in an imaging study of a premalignant bronchial lesion.
 26. The method of claim 19, wherein the subject has previously been diagnosed with a lung, bronchus, head/neck, and/or esophagus cancer but has no current evidence of the cancer.
 27. The method of claim 19, wherein the subject is a current smoker or a former smoker with 20+ pack years.
 28. The method of claim 27, wherein the subject is at least 50 years old.
 29. The method of claim 19, wherein the subject has emphysema, chronic bronchitis, chronic obstructive pulmonary disease, an occupationally related asbestos disease, or a family history of lung cancer in a first degree relative.
 30. The method of claim 29, wherein the subject is at least 50 years old. 